Insecticidal proteins and methods for their use

ABSTRACT

Compositions and methods for controlling pests are provided. The methods involve transforming organisms with a nucleic acid sequence encoding an insecticidal protein. In particular, the nucleic acid sequences are useful for preparing plants and microorganisms that possess insecticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. Compositions are insecticidal nucleic acids and proteins of bacterial species. The sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest including plants, as probes for the isolation of other homologous (or partially homologous) genes. The pesticidal proteins find use in controlling, inhibiting growth or killing Lepidopteran, Coleopteran, Dipteran, fungal, Hemipteran and nematode pest populations and for producing compositions with insecticidal activity.

CROSS REFERENCE

This application is continuation of U.S. Ser. No. 15/116,740, filed onAug. 4, 2016, which is a 371 (National Stage) of PCT/US15/14824 filedFeb. 6, 2015, which claims the benefit of U.S. Provisional ApplicationNo. 61/937,295 filed Feb. 7, 2014, and U.S. Provisional Application No.62/051,720 filed Sep. 17, 2014, which are incorporated herein byreference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“6054USPCN_Sequence_Listing” created on Sep. 19, 2018, and having a sizeof 3,706 kilobytes and is filed concurrently with the specification. Thesequence listing contained in this ASCII formatted document is part ofthe specification and is herein incorporated by reference in itsentirety.

FIELD

This disclosure relates to the field of molecular biology. Provided arenovel genes that encode pesticidal proteins. These pesticidal proteinsand the nucleic acid sequences that encode them are useful in preparingpesticidal formulations and in the production of transgenicpest-resistant plants.

BACKGROUND

Biological control of insect pests of agricultural significance using amicrobial agent, such as fungi, bacteria or another species of insectaffords an environmentally friendly and commercially attractivealternative to synthetic chemical pesticides. Generally speaking, theuse of biopesticides presents a lower risk of pollution andenvironmental hazards and biopesticides provide greater targetspecificity than is characteristic of traditional broad-spectrumchemical insecticides. In addition, biopesticides often cost less toproduce and thus improve economic yield for a wide variety of crops.

Certain species of microorganisms of the genus Bacillus are known topossess pesticidal activity against a range of insect pests includingLepidoptera, Diptera, Coleoptera, Hemiptera and others. Bacillusthuringiensis (Bt) and Bacillus popilliae are among the most successfulbiocontrol agents discovered to date. Insect pathogenicity has also beenattributed to strains of B. larvae, B. lentimorbus, B. sphaericus and B.cereus. Microbial insecticides, particularly those obtained fromBacillus strains, have played an important role in agriculture asalternatives to chemical pest control.

Crop plants have been developed with enhanced insect resistance bygenetically engineering crop plants to produce pesticidal proteins fromBacillus. For example, corn and cotton plants have been geneticallyengineered to produce pesticidal proteins isolated from strains of Bt.These genetically engineered crops are now widely used in agricultureand have provided the farmer with an environmentally friendlyalternative to traditional insect-control methods. While they haveproven to be very successful commercially, these genetically engineered,insect-resistant crop plants provide resistance to only a narrow rangeof the economically important insect pests. In some cases, insects candevelop resistance to different insecticidal compounds, which raises theneed to identify alternative biological control agents for pest control.

Accordingly, there remains a need for new pesticidal proteins withdifferent ranges of insecticidal activity against insect pests, e.g.,insecticidal proteins which are active against a variety of insects inthe order Lepidoptera and/or the order Coleoptera including but notlimited to insect pests that have developed resistance to existinginsecticides.

SUMMARY

Compositions and methods for conferring pesticidal activity to bacteria,plants, plant cells, tissues and seeds are provided. Compositionsinclude nucleic acid molecules encoding sequences for pesticidal andinsecticidal polypeptides, vectors comprising those nucleic acidmolecules, and host cells comprising the vectors. Compositions alsoinclude the pesticidal polypeptide sequences and antibodies to thosepolypeptides. The nucleic acid sequences can be used in DNA constructsor expression cassettes for transformation and expression in organisms,including microorganisms and plants. The nucleotide or amino acidsequences may be synthetic sequences that have been designed forexpression in an organism including, but not limited to, a microorganismor a plant. Compositions also comprise transformed bacteria, plants,plant cells, tissues and seeds.

In particular, isolated or recombinant nucleic acid molecules areprovided encoding Pteridophyta Insecticidal Protein-83 (PtIP-83)polypeptides including amino acid substitutions, deletions, insertions,fragments thereof. Additionally, amino acid sequences corresponding tothe PtIP-83 polypeptides are encompassed. Provided are isolated orrecombinant nucleic acid molecules capable of encoding PtIP-83polypeptides of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ IDNO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758,SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ IDNO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767,SEQ ID NO: 768, and SEQ ID NO: 769, as well as amino acid substitutions,deletions, insertions, fragments thereof, and combinations thereof.Nucleic acid sequences that are complementary to a nucleic acid sequenceof the embodiments or that hybridize to a sequence of the embodimentsare also encompassed. Also provided are isolated or recombinant PtIP-83polypeptides of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ IDNO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758,SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ IDNO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767,SEQ ID NO: 768, and SEQ ID NO: 769, as well as amino acid substitutions,deletions, insertions, fragments thereof and combinations thereof.

Methods are provided for producing the polypeptides and for using thosepolypeptides for controlling or killing a Lepidopteran, Coleopteran,nematode, fungi, and/or Dipteran pests. The transgenic plants of theembodiments express one or more of the pesticidal sequences disclosedherein. In various embodiments, the transgenic plant further comprisesone or more additional genes for insect resistance, for example, one ormore additional genes for controlling Coleopteran, Lepidopteran,Hemipteran or nematode pests. It will be understood by one of skill inthe art that the transgenic plant may comprise any gene imparting anagronomic trait of interest.

Methods for detecting the nucleic acids and polypeptides of theembodiments in a sample are also included. A kit for detecting thepresence of a PtIP-83 polypeptide or detecting the presence of apolynucleotide encoding a PtIP-83 polypeptide in a sample is provided.The kit may be provided along with all reagents and control samplesnecessary for carrying out a method for detecting the intended agent, aswell as instructions for use.

The compositions and methods of the embodiments are useful for theproduction of organisms with enhanced pest resistance or tolerance.These organisms and compositions comprising the organisms are desirablefor agricultural purposes. The compositions of the embodiments are alsouseful for generating altered or improved proteins that have pesticidalactivity or for detecting the presence of PtIP-83 polypeptides.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 the phylogeny of ferns based on the classification for extantferns by A. R. Smith et al, TAXON, 55:705-731 (2006).

FIG. 2a-2j shows an alignment of the amino acid sequences of PtIP-83Aa(SEQ ID NO: 1), PtIP-83Ca (SEQ ID NO: 5), PtIP-83Cb (SEQ ID NO: 7),PtIP-83Cc (SEQ ID NO: 9), PtIP-83Cd (SEQ ID NO: 11), PtIP-83Ce (SEQ IDNO: 13), PtIP-83Cf (SEQ ID NO: 15), and PtIP-83Fa (SEQ ID NO: 3); analignment of the secondary structure prediction, by the PSIPRED, topranked secondary structure prediction method, for PtIP-83Aa (SEQ IDNO: 1) and PtIP-83Fa (SEQ ID NO: 3); and the locations of the amino acidsequence MOTIFs, as predicted by MEME motif analysis, relative toPtIP-83Aa (SEQ ID NO: 1). A “H” indicates a predicted helical structure,an “E” indicates a PtIP-beta strand structure, and a “C” indicates apredicted coil structure.

FIG. 3a-3b shows a sequence alignment between PtIP-83Aa (SEQ ID NO: 1)and PtIP-50Aa (SEQ ID NO: 34). The crossover points in the PtIP-83Aa/PtIP-50Aa chimeras indicated in Table 13 are indicated by an arrow(↓) above the amino acid.

FIG. 4a-4c shows an amino acid sequence alignment of PtIP-83Aa (SEQ IDNO: 1), PtIP-83Fa (SEQ ID NO: 3), PtIP-50Aa (SEQ ID NO: 34), PtIP-50Ba(SEQ ID NO: 35), and PtIP-50Bb (SEQ ID NO: 36). The conserved sequencemotifs identified are indicated and the amino acid sequence of themotifs in PtIP-83Aa (SEQ ID NO: 1) are underlined.

FIG. 5a-5e shows an amino acid sequence alignment of PtIP-83Aa (SEQ IDNO: 1), PtIP-83Ca (SEQ ID NO: 5), PtIP-83Cb (SEQ ID NO: 7), PtIP-83Cc(SEQ ID NO: 9), PtIP-83Cd (SEQ ID NO: 11), PtIP-83Ce (SEQ ID NO: 13),PtIP-83Cf (SEQ ID NO: 15), PtIP-83Cg (SEQ ID NO: 17), and PtIP-83 Da(SEQ ID NO: 19). The sequence diversity is highlighted.

DETAILED DESCRIPTION

It is to be understood that this disclosure is not limited to theparticular methodology, protocols, cell lines, genera, and reagentsdescribed, as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentdisclosure.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisdisclosure belongs unless clearly indicated otherwise.

The present disclosure is drawn to compositions and methods forcontrolling pests. The methods involve transforming organisms withnucleic acid sequences encoding PtIP-83 polypeptides. In particular, thenucleic acid sequences of the embodiments are useful for preparingplants and microorganisms that possess pesticidal activity. Thus,transformed bacteria, plants, plant cells, plant tissues and seeds areprovided. The compositions are pesticidal nucleic acids and proteins ofbacterial species. The nucleic acid sequences find use in theconstruction of expression vectors for subsequent transformation intoorganisms of interest, as probes for the isolation of other homologous(or partially homologous) genes, and for the generation of alteredPtIP-83 polypeptides by methods known in the art, such as site directedmutagenesis, domain swapping or DNA shuffling. The PtIP-83 find use incontrolling or killing Lepidopteran, Coleopteran, Dipteran, fungal,Hemipteran and nematode pest populations and for producing compositionswith pesticidal activity. Insect pests of interest include, but are notlimited to, Lepidoptera species including but not limited to: CornEarworm, (CEW) (Helicoverpa zea), European Corn Borer (ECB) (Ostrinianubilalis), diamond-back moth, e.g., Helicoverpa zea Boddie; soybeanlooper, e.g., Pseudoplusia includens Walker; and velvet bean caterpillare.g., Anticarsia gemmatalis Hübner and Coleoptera species including butnot limited to Western corn rootworm (Diabrotica virgifera)—WCRW,Southern corn rootworm (Diabrotica undecimpunctata howardi)—SCRW, andNorthern corn rootworm (Diabrotica barberi)—NCRW.

By “pesticidal toxin” or “pesticidal protein” is used herein to refer toa toxin that has toxic activity against one or more pests, including,but not limited to, members of the Lepidoptera, Diptera, Hemiptera andColeoptera orders or the Nematoda phylum or a protein that has homologyto such a protein. Pesticidal proteins have been purified from organismsincluding, for example, Bacillus sp., Pseudomonas sp., Photorhabdus sp.,Xenorhabdus sp., Clostridium bifermentans and Paenibacillus popilliae.Pesticidal proteins include but are not limited to: insecticidalproteins from Pseudomonas sp. such as PSEEN3174 (Monalysin; (2011) PLoSPathogens 7:1-13); from Pseudomonas protegens strain CHAO and Pf-5(previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology10:2368-2386; GenBank Accession No. EU400157); from PseudomonasTaiwanensis (Liu, et al., (2010) J. Agric. Food Chem., 58:12343-12349)and from Pseudomonas pseudoalcligenes (Zhang, et al., (2009) Annals ofMicrobiology 59:45-50 and Li, et al., (2007) Plant Cell Tiss. OrganCult. 89:159-168); insecticidal proteins from Photorhabdus sp. andXenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxicology Journal,3:101-118 and Morgan, et al., (2001) Applied and Envir. Micro.67:2062-2069); U.S. Pat. Nos. 6,048,838, and 6,379,946; a PIP-1polypeptide of US Patent Publication US20140007292; an AfIP-1A and/orAfIP-1B polypeptide of US Patent Publication US20140033361; a PHI-4polypeptide of US patent Publication US20140274885 and PCT PatentPublication WO2014/150914; a PIP-47 polypeptide of PCT Serial NumberPCT/US14/51063, a PIP-72 polypeptide of PCT Serial NumberPCT/US14/55128, and δ-endotoxins including, but not limited to, theCry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11,Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21,Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry 28, Cry 29, Cry 30, Cry31,Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38, Cry39, Cry40, Cry41,Cry42, Cry43, Cry44, Cry45, Cry 46, Cry47, Cry49, Cry50, Cry51, Cry52,Cry53, Cry 54, Cry55, Cry56, Cry57, Cry58, Cry59, Cry60, Cry61, Cry62,Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70, Cry71, and Cry72 classes of δ-endotoxin genes and the B. thuringiensis cytolytic cyt1and cyt2 genes. Members of these classes of B. thuringiensisinsecticidal proteins include, but are not limited to Cry1Aa1 (Accession# AAA22353); Cry1Aa2 (Accession # Accession # AAA22552); Cry1Aa3(Accession # BAA00257); Cry1Aa4 (Accession # CAA31886); Cry1Aa5(Accession # BAA04468); Cry1Aa6 (Accession # AAA86265); Cry1Aa7(Accession # AAD46139); Cry1Aa8 (Accession # I26149); Cry1Aa9 (Accession# BAA77213); Cry1Aa10 (Accession # AAD55382); Cry1Aa11 (Accession #CAA70856); Cry1Aa12 (Accession # AAP80146); Cry1Aa13 (Accession #AAM44305); Cry1Aa14 (Accession # AAP40639); Cry1Aa15 (Accession #AAY66993); Cry1Aa16 (Accession # HQ439776); Cry1Aa17 (Accession #HQ439788); Cry1Aa18 (Accession # HQ439790); Cry1Aa19 (Accession #HQ685121); Cry1Aa20 (Accession # JF340156); Cry1Aa21 (Accession #JN651496); Cry1Aa22 (Accession # KC158223); Cry1Ab1 (Accession #AAA22330); Cry1Ab2 (Accession # AAA22613); Cry1Ab3 (Accession #AAA22561); Cry1Ab4 (Accession # BAA00071); Cry1Ab5 (Accession #CAA28405); Cry1Ab6 (Accession # AAA22420); Cry1Ab7 (Accession #CAA31620); Cry1Ab8 (Accession # AAA22551); Cry1Ab9 (Accession #CAA38701); Cry1Ab10 (Accession # A29125); Cry1Ab11 (Accession # I12419);Cry1Ab12 (Accession # AAC64003); Cry1Ab13 (Accession # AAN76494);Cry1Ab14 (Accession # AAG16877); Cry1Ab15 (Accession # AA013302);Cry1Ab16 (Accession # AAK55546); Cry1Ab17 (Accession # AAT46415);Cry1Ab18 (Accession # AAQ88259); Cry1Ab19 (Accession # AAW31761);Cry1Ab20 (Accession # ABB72460); Cry1Ab21 (Accession # ABS18384);Cry1Ab22 (Accession #ABW87320); Cry1Ab23 (Accession # HQ439777);Cry1Ab24 (Accession # HQ439778); Cry1Ab25 (Accession # HQ685122);Cry1Ab26 (Accession # HQ847729); Cry1Ab27 (Accession # JN135249);Cry1Ab28 (Accession # JN135250); Cry1Ab29 (Accession # JN135251);Cry1Ab30 (Accession # JN135252); Cry1Ab31 (Accession # JN135253);Cry1Ab32 (Accession # JN135254); Cry1Ab33 (Accession # AAS93798);Cry1Ab34 (Accession # KC156668); Cry1Ab-like (Accession # AAK14336);Cry1Ab-like (Accession # AAK14337); Cry1Ab-like (Accession # AAK14338);Cry1Ab-like (Accession # ABG88858); Cry1Ac1 (Accession # AAA22331);Cry1Ac2 (Accession # AAA22338); Cry1Ac3 (Accession # CAA38098); Cry1Ac4(Accession # AAA73077); Cry1Ac5 (Accession # AAA22339); Cry1Ac6(Accession # AAA86266); Cry1Ac7 (Accession # AAB46989); Cry1Ac8(Accession # AAC44841); Cry1Ac9 (Accession # AAB49768); Cry1Ac10(Accession # CAA05505); Cry1Ac11 (Accession # CAA10270); Cry1Ac12(Accession # I12418); Cry1Ac13 (Accession # AAD38701); Cry1Ac14(Accession # AAQ06607); Cry1Ac15 (Accession # AAN07788); Cry1Ac16(Accession # AAU87037); Cry1Ac17 (Accession # AAX18704); Cry1Ac18(Accession # AAY88347); Cry1Ac19 (Accession # ABD37053); Cry1Ac20(Accession # ABB89046); Cry1Ac21 (Accession # AAY66992); Cry1Ac22(Accession # ABZ01836); Cry1Ac23 (Accession # CAQ30431); Cry1Ac24(Accession # ABL01535); Cry1Ac25 (Accession # FJ513324); Cry1Ac26(Accession # FJ617446); Cry1Ac27 (Accession # FJ617447); Cry1Ac28(Accession # ACM90319); Cry1Ac29 (Accession # DQ438941); Cry1Ac30(Accession # GQ227507); Cry1Ac31 (Accession # GU446674); Cry1Ac32(Accession # HM061081); Cry1Ac33 (Accession # GQ866913); Cry1Ac34(Accession # HQ230364); Cry1Ac35 (Accession # JF340157); Cry1Ac36(Accession # JN387137); Cry1Ac37 (Accession # JQ317685); Cry1Ad1(Accession # AAA22340); Cry1Ad2 (Accession # CAA01880); Cry1Ae1(Accession # AAA22410); Cry1Af1 (Accession # AAB82749); Cry1Ag1(Accession # AAD46137); Cry1Ah1 (Accession # AAQ14326); Cry1Ah2(Accession # ABB76664); Cry1Ah3 (Accession # HQ439779); Cry1Ai1(Accession # AA039719); Cry1Ai2 (Accession # HQ439780); Cry1A-like(Accession # AAK14339); Cry1Ba1 (Accession # CAA29898); Cry1Ba2(Accession # CAA65003); Cry1Ba3 (Accession # AAK63251); Cry1Ba4(Accession # AAK51084); Cry1Ba5 (Accession # AB020894); Cry1Ba6(Accession # ABL60921); Cry1Ba7 (Accession # HQ439781); Cry1Bb1(Accession # AAA22344); Cry1Bb2 (Accession # HQ439782); Cry1Bc1(Accession # CAA86568); Cry1Bd1 (Accession # AAD10292); Cry1Bd2(Accession # AAM93496); Cry1Be1 (Accession # AAC32850); Cry1Be2(Accession # AAQ52387); Cry1Be3 (Accession # ACV96720); Cry1Be4(Accession # HM070026); Cry1Bf1 (Accession # CAC50778); Cry1Bf2(Accession # AAQ52380); Cry1Bg1 (Accession # AA039720); Cry1Bh1(Accession # HQ589331); Cry1Bi1 (Accession # KC156700); Cry1Ca1(Accession # CAA30396); Cry1Ca2 (Accession # CAA31951); Cry1Ca3(Accession # AAA22343); Cry1Ca4 (Accession # CAA01886); Cry1Ca5(Accession # CAA65457); Cry1Ca6 [1] (Accession # AAF37224); Cry1Ca7(Accession # AAG50438); Cry1Ca8 (Accession # AAM00264); Cry1Ca9(Accession # AAL79362); Cry1Ca10 (Accession # AAN16462); Cry1Ca11(Accession # AAX53094); Cry1Ca12 (Accession # HM070027); Cry1Ca13(Accession # HQ412621); Cry1Ca14 (Accession # JN651493); Cry1Cb1(Accession # M97880); Cry1Cb2 (Accession # AAG35409); Cry1Cb3 (Accession# ACD50894); Cry1Cb-like (Accession # AAX63901); Cry1Da1 (Accession #CAA38099); Cry1Da2 (Accession #176415); Cry1Da3 (Accession # HQ439784);Cry1Db (Accession # CAA80234); Cry1Db2 (Accession # AAK48937); Cry1Dc1(Accession # ABK35074); Cry1Ea1 (Accession # CAA37933); Cry1Ea2(Accession # CAA39609); Cry1Ea3 (Accession # AAA22345); Cry1Ea4(Accession # AADO4732); Cry1Ea5 (Accession # A15535); Cry1Ea6 (Accession# AAL50330); Cry1Ea7 (Accession # AAW72936); Cry1Ea8 (Accession #ABX11258); Cry1Ea9 (Accession # HQ439785); Cry1Ea10 (Accession #ADR00398); Cry1Ea11 (Accession # JQ652456); Cry1Eb1 (Accession #AAA22346); Cry1Fa1 (Accession # AAA22348); Cry1Fa2 (Accession #AAA22347); Cry1Fa3 (Accession # HM070028); Cry1Fa4 (Accession #HM439638); Cry1Fb1 (Accession # CAA80235); Cry1Fb2 (Accession #BAA25298); Cry1Fb3 (Accession # AAF21767); Cry1Fb4 (Accession #AAC10641); Cry1Fb5 (Accession # AAO13295); Cry1Fb6 (Accession #ACD50892); Cry1Fb7 (Accession # ACD50893); Cry1Ga1 (Accession #CAA80233); Cry1Ga2 (Accession # CAA70506); Cry1Gb1 (Accession #AAD10291); Cry1Gb2 (Accession # AA013756); Cry1Gc1 (Accession #AAQ52381); Cry1Ha1 (Accession # CAA80236); Cry1Hb1 (Accession #AAA79694); Cry1Hb2 (Accession # HQ439786); Cry1H-like (Accession #AAF01213); Cry1Ia1 (Accession # CAA44633); Cry1Ia2 (Accession #AAA22354); Cry1Ia3 (Accession # AAC36999); Cry1Ia4 (Accession #AAB00958); Cry1Ia5 (Accession # CAA70124); Cry1Ia6 (Accession #AAC26910); Cry1Ia7 (Accession # AAM73516); Cry1Ia8 (Accession #AAK66742); Cry1Ia9 (Accession # AAQ08616); Cry1Ia10 (Accession #AAP86782); Cry1Ia11 (Accession # CAC85964); Cry1Ia12 (Accession #AAV53390); Cry1Ia13 (Accession # ABF83202); Cry1Ia14 (Accession #ACG63871); Cry1Ia15 (Accession # FJ617445); Cry1Ia16 (Accession #FJ617448); Cry1Ia17 (Accession # GU989199); Cry1Ia18 (Accession #ADK23801); Cry1Ia19 (Accession # HQ439787); Cry1Ia20 (Accession #JQ228426); Cry1Ia21 (Accession # JQ228424); Cry1Ia22 (Accession #JQ228427); Cry1Ia23 (Accession # JQ228428); Cry1Ia24 (Accession #JQ228429); Cry1Ia25 (Accession # JQ228430); Cry1Ia26 (Accession #JQ228431); Cry1Ia27 (Accession #JQ228432); Cry1Ia28 (Accession #JQ228433); Cry1Ia29 (Accession # JQ228434); Cry1Ia30 (Accession #JQ317686); Cry1Ia31 (Accession # JX944038); Cry1Ia32 (Accession #JX944039); Cry1Ia33 (Accession # JX944040); Cry1Ib1 (Accession #AAA82114); Cry1Ib2 (Accession # ABW88019); Cry1Ib3 (Accession #ACD75515); Cry1Ib4 (Accession # HM051227); Cry1Ib5 (Accession #HM070028); Cry1Ib6 (Accession # ADK38579); Cry1Ib7 (Accession #JN571740); Cry1Ib8 (Accession # JN675714); Cry1Ib9 (Accession #JN675715); Cry1Ib10 (Accession # JN675716); Cry1Ib11 (Accession #JQ228423); Cry1Ic1 (Accession # AAC62933); Cry1Ic2 (Accession #AAE71691); Cry1Id1 (Accession # AAD44366); Cry1Id2 (Accession #JQ228422); Cry1Ie1 (Accession # AAG43526); Cry1Ie2 (Accession #HM439636); Cry1Ie3 (Accession # KC156647); Cry1Ie4 (Accession #KC156681); Cry1If1 (Accession # AAQ52382); Cry1Ig1 (Accession #KC156701); Cry1I-like (Accession # AAC31094); Cry1I-like (Accession #ABG88859); Cry1Ja1 (Accession # AAA22341); Cry1Ja2 (Accession #HM070030); Cry1Ja3 (Accession # JQ228425); Cry1Jb1 (Accession #AAA98959); Cry1Jc1 (Accession # AAC31092); Cry1Jc2 (Accession #AAQ52372); Cry1Jd1 (Accession # CAC50779); Cry1Ka1 (Accession #AAB00376); Cry1Ka2 (Accession # HQ439783); Cry1La1 (Accession #AAS60191); Cry1La2 (Accession # HM070031); Cry1Ma1 (Accession #FJ884067); Cry1Ma2 (Accession # KC156659); Cry1Na1 (Accession #KC156648); Cry1Nb1 (Accession # KC156678); Cry1-like (Accession #AAC31091); Cry2Aa1 (Accession # AAA22335); Cry2Aa2 (Accession #AAA83516); Cry2Aa3 (Accession # D86064); Cry2Aa4 (Accession # AAC04867);Cry2Aa5 (Accession # CAA10671); Cry2Aa6 (Accession # CAA10672); Cry2Aa7(Accession # CAA10670); Cry2Aa8 (Accession # AA013734); Cry2Aa9(Accession # AA013750); Cry2Aa10 (Accession # AAQ04263); Cry2Aa11(Accession # AAQ52384); Cry2Aa12 (Accession # AB183671); Cry2Aa13(Accession # ABL01536); Cry2Aa14 (Accession # ACF04939); Cry2Aa15(Accession # JN426947); Cry2Ab1 (Accession # AAA22342); Cry2Ab2(Accession # CAA39075); Cry2Ab3 (Accession # AAG36762); Cry2Ab4(Accession # AAO13296); Cry2Ab5 (Accession # AAQ04609); Cry2Ab6(Accession # AAP59457); Cry2Ab7 (Accession # AAZ66347); Cry2Ab8(Accession # ABC95996); Cry2Ab9 (Accession # ABC74968); Cry2Ab10(Accession # EF157306); Cry2Ab11 (Accession # CAM84575); Cry2Ab12(Accession # ABM21764); Cry2Ab13 (Accession # ACG76120); Cry2Ab14(Accession # ACG76121); Cry2Ab15 (Accession # HM037126); Cry2Ab16(Accession # GQ866914); Cry2Ab17 (Accession # HQ439789); Cry2Ab18(Accession # JN135255); Cry2Ab19 (Accession # JN135256); Cry2Ab20(Accession # JN135257); Cry2Ab21 (Accession # JN135258); Cry2Ab22(Accession # JN135259); Cry2Ab23 (Accession # JN135260); Cry2Ab24(Accession # JN135261); Cry2Ab25 (Accession # JN415485); Cry2Ab26(Accession # JN426946); Cry2Ab27 (Accession # JN415764); Cry2Ab28(Accession # JN651494); Cry2Ac1 (Accession # CAA40536); Cry2Ac2(Accession # AAG35410); Cry2Ac3 (Accession # AAQ52385); Cry2Ac4(Accession # ABC95997); Cry2Ac5 (Accession # ABC74969); Cry2Ac6(Accession # ABC74793); Cry2Ac7 (Accession # CAL18690); Cry2Ac8(Accession # CAM09325); Cry2Ac9 (Accession # CAM09326); Cry2Ac10(Accession # ABN15104); Cry2Ac11 (Accession # CAM83895); Cry2Ac12(Accession # CAM83896); Cry2Ad1 (Accession # AAF09583); Cry2Ad2(Accession # ABC86927); Cry2Ad3 (Accession # CAK29504); Cry2Ad4(Accession # CAM32331); Cry2Ad5 (Accession # CA078739); Cry2Ae1(Accession # AAQ52362); Cry2Af1 (Accession # AB030519); Cry2Af2(Accession # GQ866915); Cry2Ag1 (Accession # ACH91610); Cry2Ah1(Accession # EU939453); Cry2Ah2 (Accession # ACL80665); Cry2Ah3(Accession # GU073380); Cry2Ah4 (Accession # KC156702); Cry2Ai1(Accession # FJ788388); Cry2Aj (Accession #); Cry2Ak1 (Accession #KC156660); Cry2Ba1 (Accession # KC156658); Cry3Aa1 (Accession #AAA22336); Cry3Aa2 (Accession # AAA22541); Cry3Aa3 (Accession #CAA68482); Cry3Aa4 (Accession # AAA22542); Cry3Aa5 (Accession #AAA50255); Cry3Aa6 (Accession # AAC43266); Cry3Aa7 (Accession #CAB41411); Cry3Aa8 (Accession # AAS79487); Cry3Aa9 (Accession #AAW05659); Cry3Aa10 (Accession # AAU29411); Cry3Aa11 (Accession #AAW82872); Cry3Aa12 (Accession # ABY49136); Cry3Ba1 (Accession #CAA34983); Cry3Ba2 (Accession # CAA00645); Cry3Ba3 (Accession #JQ397327); Cry3Bb1 (Accession # AAA22334); Cry3Bb2 (Accession #AAA74198); Cry3Bb3 (Accession # I15475); Cry3Ca1 (Accession # CAA42469);Cry4Aa1 (Accession # CAA68485); Cry4Aa2 (Accession # BAA00179); Cry4Aa3(Accession # CAD30148); Cry4Aa4 (Accession # AFB18317); Cry4A-like(Accession # AAY96321); Cry4Ba1 (Accession # CAA30312); Cry4Ba2(Accession # CAA30114); Cry4Ba3 (Accession # AAA22337); Cry4Ba4(Accession # BAA00178); Cry4Ba5 (Accession # CAD30095); Cry4Ba-like(Accession # ABC47686); Cry4Ca1 (Accession # EU646202); Cry4Cb1(Accession # FJ403208); Cry4Cb2 (Accession # FJ597622); Cry4Cc1(Accession # FJ403207); Cry5Aa1 (Accession # AAA67694); Cry5Ab1(Accession # AAA67693); Cry5Ac1 (Accession # I34543); Cry5Ad1 (Accession# ABQ82087); Cry5Ba1 (Accession # AAA68598); Cry5Ba2 (Accession #ABW88931); Cry5Ba3 (Accession # AFJ04417); Cry5Ca1 (Accession #HM461869); Cry5Ca2 (Accession # ZP_04123426); Cry5Da1 (Accession #HM461870); Cry5Da2 (Accession # ZP_04123980); Cry5Ea1 (Accession #HM485580); Cry5Ea2 (Accession # ZP_04124038); Cry6Aa1 (Accession #AAA22357); Cry6Aa2 (Accession # AAM46849); Cry6Aa3 (Accession #ABH03377); Cry6Ba1 (Accession # AAA22358); Cry7Aa1 (Accession #AAA22351); Cry7Ab1 (Accession #AAA21120); Cry7Ab2 (Accession #AAA21121); Cry7Ab3 (Accession # ABX24522); Cry7Ab4 (Accession #EU380678); Cry7Ab5 (Accession # ABX79555); Cry7Ab6 (Accession #AC144005); Cry7Ab7 (Accession # ADB89216); Cry7Ab8 (Accession #GU145299); Cry7Ab9 (Accession # ADD92572); Cry7Ba1 (Accession #ABB70817); Cry7Bb1 (Accession # KC156653); Cry7Ca1 (Accession #ABR67863); Cry7Cb1 (Accession # KC156698); Cry7Da1 (Accession #ACQ99547); Cry7Da2 (Accession # HM572236); Cry7Da3 (Accession #KC156679); Cry7Ea1 (Accession # HM035086); Cry7Ea2 (Accession #HM132124); Cry7Ea3 (Accession # EEM19403); Cry7Fa1 (Accession #HM035088); Cry7Fa2 (Accession # EEM19090); Cry7Fb1 (Accession #HM572235); Cry7Fb2 (Accession # KC156682); Cry7Ga1 (Accession #HM572237); Cry7Ga2 (Accession # KC156669); Cry7Gb1 (Accession #KC156650); Cry7Gc1 (Accession # KC156654); Cry7Gd1 (Accession #KC156697); Cry7Ha1 (Accession # KC156651); Cry7Ia1 (Accession #KC156665); Cry7Ja1 (Accession # KC156671); Cry7Ka1 (Accession #KC156680); Cry7Kb1 (Accession # BAM99306); Cry7La1 (Accession #BAM99307); Cry8Aa1 (Accession # AAA21117); Cry8Ab1 (Accession #EU044830); Cry8Ac1 (Accession # KC156662); Cry8Ad1 (Accession #KC156684); Cry8Ba1 (Accession # AAA21118); Cry8Bb1 (Accession #CAD57542); Cry8Bc1 (Accession # CAD57543); Cry8Ca1 (Accession #AAA21119); Cry8Ca2 (Accession # AAR98783); Cry8Ca3 (Accession #EU625349); Cry8Ca4 (Accession # ADB54826); Cry8Da1 (Accession #BAC07226); Cry8Da2 (Accession # BD133574); Cry8Da3 (Accession #BD133575); Cry8Db1 (Accession # BAF93483); Cry8Ea1 (Accession #AAQ73470); Cry8Ea2 (Accession # EU047597); Cry8Ea3 (Accession #KC855216); Cry8Fa1 (Accession # AAT48690); Cry8Fa2 (Accession #HQ174208); Cry8Fa3 (Accession # AFH78109); Cry8Ga1 (Accession #AAT46073); Cry8Ga2 (Accession # ABC42043); Cry8Ga3 (Accession #FJ198072); Cry8Ha1 (Accession # AAW81032); Cry8Ia1 (Accession #EU381044); Cry8Ia2 (Accession # GU073381); Cry8Ia3 (Accession #HM044664); Cry8Ia4 (Accession # KC156674); Cry8Ib1 (Accession #GU325772); Cry8Ib2 (Accession # KC156677); Cry8Ja1 (Accession #EU625348); Cry8Ka1 (Accession # FJ422558); Cry8Ka2 (Accession #ACN87262); Cry8Kb1 (Accession # HM123758); Cry8Kb2 (Accession #KC156675); Cry8La1 (Accession # GU325771); Cry8Ma1 (Accession #HM044665); Cry8Ma2 (Accession # EEM86551); Cry8Ma3 (Accession #HM210574); Cry8Na1 (Accession # HM640939); Cry8Pa1 (Accession #HQ388415); Cry8Qa1 (Accession # HQ441166); Cry8Qa2 (Accession #KC152468); Cry8Ra1 (Accession # AFP87548); Cry8Sa1 (Accession #JQ740599); Cry8Ta1 (Accession # KC156673); Cry8-like (Accession #FJ770571); Cry8-like (Accession # ABS53003); Cry9Aa1 (Accession #CAA41122); Cry9Aa2 (Accession # CAA41425); Cry9Aa3 (Accession#GQ249293); Cry9Aa4 (Accession # GQ249294); Cry9Aa5 (Accession #JX174110); Cry9Aa like (Accession # AAQ52376); Cry9Ba1 (Accession #CAA52927); Cry9Ba2 (Accession # GU299522); Cry9Bb1 (Accession #AAV28716); Cry9Ca1 (Accession # CAA85764); Cry9Ca2 (Accession #AAQ52375); Cry9Da1 (Accession # BAA19948); Cry9Da2 (Accession #AAB97923); Cry9Da3 (Accession # GQ249293); Cry9Da4 (Accession #GQ249297); Cry9Db1 (Accession # AAX78439); Cry9Dc1 (Accession #KC156683); Cry9Ea1 (Accession # BAA34908); Cry9Ea2 (Accession #AAO12908); Cry9Ea3 (Accession # ABM21765); Cry9Ea4 (Accession #ACE88267); Cry9Ea5 (Accession # ACF04743); Cry9Ea6 (Accession #ACG63872); Cry9Ea7 (Accession # FJ380927); Cry9Ea8 (Accession #GQ249292); Cry9Ea9 (Accession # JN651495); Cry9Eb1 (Accession #CAC50780); Cry9Eb2 (Accession # GQ249298); Cry9Eb3 (Accession #KC156646); Cry9Ec1 (Accession # AAC63366); Cry9Ed1 (Accession #AAX78440); Cry9Ee1 (Accession # GQ249296); Cry9Ee2 (Accession #KC156664); Cry9Fa1 (Accession # KC156692); Cry9Ga1 (Accession #KC156699); Cry9-like (Accession # AAC63366); Cry10Aa1 (Accession #AAA22614); Cry10Aa2 (Accession # E00614); Cry10Aa3 (Accession #CAD30098); Cry10Aa4 (Accession # AFB18318); Cry10A-like (Accession #DQ167578); Cry11Aa1 (Accession # AAA22352); Cry11Aa2 (Accession #AAA22611); Cry11Aa3 (Accession # CAD30081); Cry11Aa4 (Accession #AFB18319); Cry11Aa-like (Accession # DQ166531); Cry11Ba1 (Accession #CAA60504); Cry11Bb1 (Accession # AAC97162); Cry11Bb2 (Accession #HM068615); Cry12Aa1 (Accession # AAA22355); Cry13Aa1 (Accession #AAA22356); Cry14Aa1 (Accession # AAA21516); Cry14Ab1 (Accession #KC156652); Cry15Aa1 (Accession # AAA22333); Cry16Aa1 (Accession #CAA63860); Cry17Aa1 (Accession # CAA67841); Cry18Aa1 (Accession #CAA67506); Cry18Ba1 (Accession # AAF89667); Cry18Ca1 (Accession #AAF89668); Cry19Aa1 (Accession # CAA68875); Cry19Ba1 (Accession #BAA32397); Cry19Ca1 (Accession # AFM37572); Cry20Aa1 (Accession #AAB93476); Cry20Ba1 (Accession # ACS93601); Cry20Ba2 (Accession #KC156694); Cry20-like (Accession # GQ144333); Cry21Aa1 (Accession #I32932); Cry21Aa2 (Accession # I66477); Cry21Ba1 (Accession # BAC06484);Cry21Ca1 (Accession # JF521577); Cry21Ca2 (Accession # KC156687);Cry21Da1 (Accession # JF521578); Cry22Aa1 (Accession # I34547); Cry22Aa2(Accession # CAD43579); Cry22Aa3 (Accession # ACD93211); Cry22Ab1(Accession # AAK50456); Cry22Ab2 (Accession # CAD43577); Cry22Ba1(Accession # CAD43578); Cry22Bb1 (Accession # KC156672); Cry23Aa1(Accession # AAF76375); Cry24Aa1 (Accession # AAC61891); Cry24Ba1(Accession # BAD32657); Cry24Ca1 (Accession # CAJ43600); Cry25Aa1(Accession # AAC61892); Cry26Aa1 (Accession # AAD25075); Cry27Aa1(Accession # BAA82796); Cry28Aa1 (Accession # AAD24189); Cry28Aa2(Accession # AAG00235); Cry29Aa1 (Accession # CAC80985); Cry30Aa1(Accession # CAC80986); Cry30Ba1 (Accession # BADO0052); Cry30Ca1(Accession # BAD67157); Cry30Ca2 (Accession # ACU24781); Cry30Da1(Accession # EF095955); Cry30Db1 (Accession # BAE80088); Cry30Ea1(Accession # ACC95445); Cry30Ea2 (Accession # FJ499389); Cry30Fa1(Accession # AC122625); Cry30Ga1 (Accession # ACG60020); Cry30Ga2(Accession # HQ638217); Cry31Aa1 (Accession # BAB11757); Cry31Aa2(Accession # AAL87458); Cry31Aa3 (Accession # BAE79808); Cry31Aa4(Accession # BAF32571); Cry31Aa5 (Accession # BAF32572); Cry31Aa6(Accession # BA144026); Cry31Ab1 (Accession # BAE79809); Cry31Ab2(Accession # BAF32570); Cry31Ac1 (Accession # BAF34368); Cry31Ac2(Accession # AB731600); Cry31Ad1 (Accession # BA144022); Cry32Aa1(Accession # AAG36711); Cry32Aa2 (Accession # GU063849); Cry32Ab1(Accession # GU063850); Cry32Ba1 (Accession # BAB78601); Cry32Ca1(Accession # BAB78602); Cry32Cb1 (Accession # KC156708); Cry32Da1(Accession # BAB78603); Cry32Ea1 (Accession # GU324274); Cry32Ea2(Accession # KC156686); Cry32Eb1 (Accession # KC156663); Cry32Fa1(Accession # KC156656); Cry32Ga1 (Accession # KC156657); Cry32Ha1(Accession # KC156661); Cry32Hb1 (Accession # KC156666); Cry32Ia1(Accession # KC156667); Cry32Ja1 (Accession # KC156685); Cry32Ka1(Accession # KC156688); Cry32La1 (Accession # KC156689); Cry32Ma1(Accession # KC156690); Cry32Mb1 (Accession # KC156704); Cry32Na1(Accession # KC156691); Cry32Oa1 (Accession # KC156703); Cry32Pa1(Accession # KC156705); Cry32Qa1 (Accession # KC156706); Cry32Ra1(Accession # KC156707); Cry32Sa1 (Accession # KC156709); Cry32Ta1(Accession # KC156710); Cry32Ua1 (Accession # KC156655); Cry33Aa1(Accession # AAL26871); Cry34Aa1 (Accession # AAG50341); Cry34Aa2(Accession # AAK64560); Cry34Aa3 (Accession # AAT29032); Cry34Aa4(Accession # AAT29030); Cry34Ab1 (Accession # AAG41671); Cry34Ac1(Accession # AAG50118); Cry34Ac2 (Accession # AAK64562); Cry34Ac3(Accession # AAT29029); Cry34Ba1 (Accession # AAK64565); Cry34Ba2(Accession # AAT29033); Cry34Ba3 (Accession # AAT29031); Cry35Aa1(Accession # AAG50342); Cry35Aa2 (Accession # AAK64561); Cry35Aa3(Accession # AAT29028); Cry35Aa4 (Accession # AAT29025); Cry35Ab1(Accession # AAG41672); Cry35Ab2 (Accession # AAK64563); Cry35Ab3(Accession # AY536891); Cry35Ac1 (Accession # AAG50117); Cry35Ba1(Accession # AAK64566); Cry35Ba2 (Accession # AAT29027); Cry35Ba3(Accession # AAT29026); Cry36Aa1 (Accession # AAK64558); Cry37Aa1(Accession # AAF76376); Cry38Aa1 (Accession # AAK64559); Cry39Aa1(Accession # BAB72016); Cry40Aa1 (Accession # BAB72018); Cry40Ba1(Accession # BAC77648); Cry40Ca1 (Accession # EU381045); Cry40Da1(Accession # ACF15199); Cry41Aa1 (Accession # BAD35157); Cry41Ab1(Accession # BAD35163); Cry41Ba1 (Accession # HM461871); Cry41Ba2(Accession # ZP_04099652); Cry42Aa1 (Accession # BAD35166); Cry43Aa1(Accession # BAD15301); Cry43Aa2 (Accession # BAD95474); Cry43Ba1(Accession # BAD15303); Cry43Ca1 (Accession # KC156676); Cry43Cb1(Accession # KC156695); Cry43Cc1 (Accession # KC156696); Cry43-like(Accession # BAD15305); Cry44Aa (Accession # BADO8532); Cry45Aa(Accession # BAD22577); Cry46Aa (Accession # BAC79010); Cry46Aa2(Accession # BAG68906); Cry46Ab (Accession # BAD35170); Cry47Aa(Accession # AAY24695); Cry48Aa (Accession # CAJ18351); Cry48Aa2(Accession # CAJ86545); Cry48Aa3 (Accession # CAJ86546); Cry48Ab(Accession # CAJ86548); Cry48Ab2 (Accession # CAJ86549); Cry49Aa(Accession # CAH56541); Cry49Aa2 (Accession # CAJ86541); Cry49Aa3(Accession # CAJ86543); Cry49Aa4 (Accession # CAJ86544); Cry49Ab1(Accession # CAJ86542); Cry50Aa1 (Accession # BAE86999); Cry50Ba1(Accession # GU446675); Cry50Ba2 (Accession # GU446676); Cry51Aa1(Accession # AB114444); Cry51Aa2 (Accession # GU570697); Cry52Aa1(Accession # EF613489); Cry52Ba1 (Accession # FJ361760); Cry53Aa1(Accession # EF633476); Cry53Ab1 (Accession # FJ361759); Cry54Aa1(Accession # ACA52194); Cry54Aa2 (Accession # GQ140349); Cry54Ba1(Accession # GU446677); Cry55Aa1 (Accession # ABW88932); Cry54Ab1(Accession # JQ916908); Cry55Aa2 (Accession # AAE33526); Cry56Aa1(Accession # ACU57499); Cry56Aa2 (Accession # GQ483512); Cry56Aa3(Accession # JX025567); Cry57Aa1 (Accession # ANC87261); Cry58Aa1(Accession # ANC87260); Cry59Ba1 (Accession # JN790647); Cry59Aa1(Accession # ACR43758); Cry60Aa1 (Accession # ACU24782); Cry60Aa2(Accession # EA057254); Cry60Aa3 (Accession # EEM99278); Cry60Ba1(Accession # GU810818); Cry60Ba2 (Accession # EA057253); Cry60Ba3(Accession # EEM99279); Cry61Aa1 (Accession # HM035087); Cry61Aa2(Accession # HM132125); Cry61Aa3 (Accession # EEM19308); Cry62Aa1(Accession # HM054509); Cry63Aa1 (Accession # BA144028); Cry64Aa1(Accession # BAJ05397); Cry65Aa1 (Accession # HM461868); Cry65Aa2(Accession # ZP_04123838); Cry66Aa1 (Accession # HM485581); Cry66Aa2(Accession # ZP_04099945); Cry67Aa1 (Accession # HM485582); Cry67Aa2(Accession # ZP_04148882); Cry68Aa1 (Accession # HQ113114); Cry69Aa1(Accession # HQ401006); Cry69Aa2 (Accession # JQ821388); Cry69Ab1(Accession # JN209957); Cry70Aa1 (Accession # JN646781); Cry70Ba1(Accession # ADO51070); Cry70Bb1 (Accession # EEL67276); Cry71Aa1(Accession # JX025568); Cry72Aa1 (Accession # JX025569); Cyt1Aa (GenBankAccession Number X03182); Cyt1Ab (GenBank Accession Number X98793);Cyt1B (GenBank Accession Number U37196); Cyt2A (GenBank Accession NumberZ14147); and Cyt2B (GenBank Accession Number U52043).

Examples of δ-endotoxins also include but are not limited to Cry1Aproteins of U.S. Pat. Nos. 5,880,275 and 7,858,849; a DIG-3 or DIG-11toxin (N-terminal deletion of α-helix 1 and/or α-helix 2 variants of cryproteins such as Cry1A, Cry3A) of U.S. Pat. Nos. 8,304,604, 8,304,605and 8,476,226; Cry1B of U.S. patent application Ser. No. 10/525,318;Cry1C of U.S. Pat. No. 6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960 and6,218,188; Cry1A/F chimeras of U.S. Pat. Nos. 7,070,982; 6,962,705 and6,713,063); a Cry2 protein such as Cry2Ab protein of U.S. Pat. No.7,064,249); a Cry3A protein including but not limited to an engineeredhybrid insecticidal protein (eHIP) created by fusing unique combinationsof variable regions and conserved blocks of at least two different Cryproteins (US Patent Application Publication Number 2010/0017914); a Cry4protein; a Cry5 protein; a Cry6 protein; Cry8 proteins of U.S. Pat. Nos.7,329,736, 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378,499 and7,462,760; a Cry9 protein such as such as members of the Cry9A, Cry9B,Cry9C, Cry9D, Cry9E and Cry9F families; a Cry15 protein of Naimov, etal., (2008) Applied and Environmental Microbiology, 74:7145-7151; aCry22, a Cry34Ab1 protein of U.S. Pat. Nos. 6,127,180, 6,624,145 and6,340,593; a CryET33 and cryET34 protein of U.S. Pat. Nos. 6,248,535,6,326,351, 6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 andCryET34 homologs of US Patent Publication Number 2006/0191034,2012/0278954, and PCT Publication Number WO 2012/139004; a Cry35Ab1protein of U.S. Pat. Nos. 6,083,499, 6,548,291 and 6,340,593; a Cry46protein, a Cry 51 protein, a Cry binary toxin; a TIC901 or relatedtoxin; TIC807 of US Patent Application Publication Number 2008/0295207;ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US2006/033867; AXMI-027, AXMI-036, and AXMI-038 of U.S. Pat. No.8,236,757; AXMI-031, AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. No.7,923,602; AXMI-018, AXMI-020 and AXMI-021 of WO 2006/083891; AXMI-010of WO 2005/038032; AXMI-003 of WO 2005/021585; AXMI-008 of US PatentApplication Publication Number 2004/0250311; AXMI-006 of US PatentApplication Publication Number 2004/0216186; AXMI-007 of US PatentApplication Publication Number 2004/0210965; AXMI-009 of US PatentApplication Number 2004/0210964; AXMI-014 of US Patent ApplicationPublication Number 2004/0197917; AXMI-004 of US Patent ApplicationPublication Number 2004/0197916; AXMI-028 and AXMI-029 of WO2006/119457; AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 andAXMI-004 of WO 2004/074462; AXMI-150 of U.S. Pat. No. 8,084,416;AXMI-205 of US Patent Application Publication Number 2011/0023184;AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI-037,AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI-063 andAXMI-064 of US Patent Application Publication Number 2011/0263488;AXMI-R1 and related proteins of US Patent Application Publication Number2010/0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z and AXMI225z of WO2011/103248; AXMI218, AXMI219, AXMI220, AXM1226, AXM1227, AXM1228,AXM1229, AXMI230 and AXMI231 of WO 2011/103247; AXMI-115, AXMI-113,AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. 8,334,431; AXMI-001,AXMI-002, AXMI-030, AXMI-035 and AXMI-045 of US Patent ApplicationPublication Number 2010/0298211; AXMI-066 and AXMI-076 of US PatentApplication Publication Number 2009/0144852; AXMI128, AXMI130, AXMI131,AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148,AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158,AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171,AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179,AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189of U.S. Pat. No. 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082, AXMI091,AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102,AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, AXMI112,AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122,AXMI123, AXMI124, AXM11257, AXM11268, AXMI127, AXMI129, AXMI164,AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of US PatentApplication Publication Number 2010/0005543, cry proteins such as Cry1Aand Cry3A having modified proteolytic sites of U.S. Pat. No. 8,319,019;a Cry1Ac, Cry2Aa and Cry1Ca toxin protein from Bacillus thuringiensisstrain VBTS 2528 of US Patent Application Publication Number2011/0064710. Other Cry proteins are well known to one skilled in theart (see, Crickmore, et al., “Bacillus thuringiensis toxin nomenclature”(2011), at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ which can beaccessed on the world-wide web using the “www” prefix). The insecticidalactivity of Cry proteins is well known to one skilled in the art (forreview, see, van Frannkenhuyzen, (2009) J. Invert. Path. 101:1-16). Theuse of Cry proteins as transgenic plant traits is well known to oneskilled in the art and Cry-transgenic plants including but not limitedto plants expressing Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F,Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1,Cry35Ab1, Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatoryapproval (see, Sanahuja, (2011) Plant Biotech Journal 9:283-300 and theCERA. (2010) GM Crop Database Center for Environmental Risk Assessment(CERA), ILSI Research Foundation, Washington D.C. atcera-gmc.org/index.php?action=gm_crop_database which can be accessed onthe world-wide web using the “www” prefix). More than one pesticidalproteins well known to one skilled in the art can also be expressed inplants such as Vip3Ab & Cry1Fa (US2012/0317682); Cry1BE & Cry1F(US2012/0311746); Cry1CA & Cry1AB (US2012/0311745); Cry1F & CryCa(US2012/0317681); Cry1DA & Cry1BE (US2012/0331590); Cry1DA & Cry1Fa(US2012/0331589); Cry1AB & Cry1BE (US2012/0324606); Cry1Fa & Cry2Aa andCry1I & Cry1E (US2012/0324605); Cry34Ab/35Ab and Cry6Aa (US20130167269);Cry34Ab/VCry35Ab & Cry3Aa (US20130167268); and Cry3A and Cry1Ab orVip3Aa (US20130116170). Pesticidal proteins also include insecticidallipases including lipid acyl hydrolases of U.S. Pat. No. 7,491,869, andcholesterol oxidases such as from Streptomyces (Purcell et al. (1993)Biochem Biophys Res Commun 15:1406-1413). Pesticidal proteins alsoinclude VIP (vegetative insecticidal proteins) toxins of U.S. Pat. Nos.5,877,012, 6,107,279 6,137,033, 7,244,820, 7,615,686, and 8,237,020 andthe like. Other VIP proteins are well known to one skilled in the art(see, lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html which can beaccessed on the world-wide web using the “www” prefix). Pesticidalproteins also include toxin complex (TC) proteins, obtainable fromorganisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, U.S.Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have “stand alone”insecticidal activity and other TC proteins enhance the activity of thestand-alone toxins produced by the same given organism. The toxicity ofa “stand-alone” TC protein (from Photorhabdus, Xenorhabdus orPaenibacillus, for example) can be enhanced by one or more TC protein“potentiators” derived from a source organism of a different genus.There are three main types of TC proteins. As referred to herein, ClassA proteins (“Protein A”) are stand-alone toxins. Class B proteins(“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity ofClass A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 andXptA2. Examples of Class B proteins are TcaC, TcdB, XptB1Xb and XptC1Wi.Examples of Class C proteins are TccC, XptC1Xb and XptB1Wi. Pesticidalproteins also include spider, snake and scorpion venom proteins.Examples of spider venom peptides include but not limited to lycotoxin-1peptides and mutants thereof (U.S. Pat. No. 8,334,366).

In some embodiments the PtIP-83 polypeptide include amino acid sequencesdeduced from the full-length nucleic acid sequences disclosed herein andamino acid sequences that are shorter than the full-length sequences,either due to the use of an alternate downstream start site or due toprocessing that produces a shorter protein having pesticidal activity.Processing may occur in the organism the protein is expressed in or inthe pest after ingestion of the protein.

Thus, provided herein are novel isolated or recombinant nucleic acidsequences that confer pesticidal activity. Also provided are the aminoacid sequences of PtIP-83 polypeptides. The protein resulting fromtranslation of these PtIP-83 polypeptide genes allows cells to controlor kill pests that ingest it.

Nucleic Acid Molecules, and Variants and Fragments Thereof

One aspect pertains to isolated or recombinant nucleic acid moleculescomprising nucleic acid sequences encoding PtIP-83 polypeptides orbiologically active portions thereof, as well as nucleic acid moleculessufficient for use as hybridization probes to identify nucleic acidmolecules encoding proteins with regions of sequence homology. As usedherein, the term “nucleic acid molecule” refers to DNA molecules (e.g.,recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) andRNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

An “isolated” nucleic acid molecule (or DNA) is used herein to refer toa nucleic acid sequence (or DNA) that is no longer in its naturalenvironment, for example in vitro. A “recombinant” nucleic acid molecule(or DNA) is used herein to refer to a nucleic acid sequence (or DNA)that is in a recombinant bacterial or plant host cell. In someembodiments, an “isolated” or “recombinant” nucleic acid is free ofsequences (preferably protein encoding sequences) that naturally flankthe nucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For purposes of the disclosure, “isolated” or“recombinant” when used to refer to nucleic acid molecules excludesisolated chromosomes. For example, in various embodiments, therecombinant nucleic acid molecule encoding PtIP-83 polypeptides cancontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kbof nucleic acid sequences that naturally flank the nucleic acid moleculein genomic DNA of the cell from which the nucleic acid is derived.

In some embodiments an isolated nucleic acid molecule encoding PtIP-83polypeptides has one or more change in the nucleic acid sequencecompared to the native or genomic nucleic acid sequence. In someembodiments the change in the native or genomic nucleic acid sequenceincludes but is not limited to: changes in the nucleic acid sequence dueto the degeneracy of the genetic code; changes in the nucleic acidsequence due to the amino acid substitution, insertion, deletion and/oraddition compared to the native or genomic sequence; removal of one ormore intron; deletion of one or more upstream or downstream regulatoryregions; and deletion of the 5′ and/or 3′ untranslated region associatedwith the genomic nucleic acid sequence. In some embodiments the nucleicacid molecule encoding a PtIP-83 polypeptide is a non-genomic sequence.

A variety of polynucleotides that encode PtIP-83 polypeptides or relatedproteins are contemplated. Such polynucleotides are useful forproduction of PtIP-83 polypeptides in host cells when operably linked tosuitable promoter, transcription termination and/or polyadenylationsequences. Such polynucleotides are also useful as probes for isolatinghomologous or substantially homologous polynucleotides that encodePtIP-83 polypeptides or related proteins.

Polynucleotides Encoding PtIP-83 Polypeptides

One source of polynucleotides that encode PtIP-83 polypeptides orrelated proteins is a fern or other primitive plant species whichcontains a PtIP-83 polynucleotide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQID NO: 717, SEQ ID NO: 738, SEQ ID NO: 739, SEQ ID NO: 740, SEQ ID NO:741, SEQ ID NO: 742, SEQ ID NO: 743, SEQ ID NO: 744, SEQ ID NO: 745, SEQID NO: 746, SEQ ID NO: 747, SEQ ID NO: 748, SEQ ID NO: 749, SEQ ID NO:750, SEQ ID NO: 751, SEQ ID NO: 752 or SEQ ID NO: 753, encoding aPtIP-83 polypeptide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO:758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO:767, SEQ ID NO: 768 or SEQ ID NO: 769. The polynucleotides of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ IDNO: 22, SEQ ID NO: 24, SEQ ID NO: 717, SEQ ID NO: 738, SEQ ID NO: 739,SEQ ID NO: 740, SEQ ID NO: 741, SEQ ID NO: 742, SEQ ID NO: 743, SEQ IDNO: 744, SEQ ID NO: 745, SEQ ID NO: 746, SEQ ID NO: 747, SEQ ID NO: 748,SEQ ID NO: 749, SEQ ID NO: 750, SEQ ID NO: 751, SEQ ID NO: 752 and SEQID NO: 753 can be used to express PtIP-83 polypeptides in bacterialhosts that include but are not limited to Agrobacterium, Bacillus,Escherichia, Salmonella, Pseudomonas and Rhizobium bacterial host cells.The polynucleotides are also useful as probes for isolating homologousor substantially homologous polynucleotides that encode PtIP-83polypeptides or related proteins. Such probes can be used to identifyhomologous or substantially homologous polynucleotides derived fromPteridophyta species.

Polynucleotides that encode PtIP-83 polypeptides can also be synthesizedde novo from a PtIP-83 polypeptide sequence. The sequence of thepolynucleotide gene can be deduced from a PtIP-83 polypeptide sequencethrough use of the genetic code. Computer programs such as“BackTranslate” (GCG™ Package, Acclerys, Inc. San Diego, Calif.) can beused to convert a peptide sequence to the corresponding nucleotidesequence encoding the peptide. Examples of PtIP-83 polypeptide sequencesthat can be used to obtain corresponding nucleotide encoding sequencesinclude, but are not limited to the PtIP-83 polypeptides of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ IDNO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755,SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ IDNO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID NO: 764,SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768, and SEQID NO: 769. Furthermore, synthetic PtIP-83 polynucleotide sequences ofthe disclosure can be designed so that they will be expressed in plants.U.S. Pat. No. 5,500,365 describes a method for synthesizing plant genesto improve the expression level of the protein encoded by thesynthesized gene. This method relates to the modification of thestructural gene sequences of the exogenous transgene, to cause them tobe more efficiently transcribed, processed, translated and expressed bythe plant. Features of genes that are expressed well in plants includeelimination of sequences that can cause undesired intron splicing orpolyadenylation in the coding region of a gene transcript whileretaining substantially the amino acid sequence of the toxic portion ofthe insecticidal protein. A similar method for obtaining enhancedexpression of transgenes in monocotyledonous plants is disclosed in U.S.Pat. No. 5,689,052.

In some embodiments the nucleic acid molecule encoding a PtIP-83polypeptide is a polynucleotide having the sequence set forth in SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQID NO: 22, SEQ ID NO: 24, SEQ ID NO: 717, SEQ ID NO: 738, SEQ ID NO:739, SEQ ID NO: 740, SEQ ID NO: 741, SEQ ID NO: 742, SEQ ID NO: 743, SEQID NO: 744, SEQ ID NO: 745, SEQ ID NO: 746, SEQ ID NO: 747, SEQ ID NO:748, SEQ ID NO: 749, SEQ ID NO: 750, SEQ ID NO: 751, SEQ ID NO: 752, SEQID NO: 753, and variants, fragments and complements thereof.“Complement” is used herein to refer to a nucleic acid sequence that issufficiently complementary to a given nucleic acid sequence such that itcan hybridize to the given nucleic acid sequence to thereby form astable duplex. “Polynucleotide sequence variants” is used herein torefer to a nucleic acid sequence that except for the degeneracy of thegenetic code encodes the same polypeptide.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is a non-genomic nucleic acid sequence. As used herein a“non-genomic nucleic acid sequence” or “non-genomic nucleic acidmolecule” or “non-genomic polynucleotide” refers to a nucleic acidmolecule that has one or more change in the nucleic acid sequencecompared to a native or genomic nucleic acid sequence. In someembodiments the change to a native or genomic nucleic acid moleculeincludes but is not limited to: changes in the nucleic acid sequence dueto the degeneracy of the genetic code; codon optimization of the nucleicacid sequence for expression in plants; changes in the nucleic acidsequence to introduce at least one amino acid substitution, insertion,deletion and/or addition compared to the native or genomic sequence;removal of one or more intron associated with the genomic nucleic acidsequence; insertion of one or more heterologous introns; deletion of oneor more upstream or downstream regulatory regions associated with thegenomic nucleic acid sequence; insertion of one or more heterologousupstream or downstream regulatory regions; deletion of the 5′ and/or 3′untranslated region associated with the genomic nucleic acid sequence;insertion of a heterologous 5′ and/or 3′ untranslated region; andmodification of a polyadenylation site. In some embodiments thenon-genomic nucleic acid molecule is a cDNA. In some embodiments thenon-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

In some embodiments the nucleic acid molecule encoding a PtIP-83polypeptide is a the non-genomic polynucleotide having a nucleotidesequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identity, to the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14,SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO: 717, SEQ ID NO: 738, SEQ ID NO: 739, SEQ ID NO: 740, SEQID NO: 741, SEQ ID NO: 742, SEQ ID NO: 743, SEQ ID NO: 744, SEQ ID NO:745, SEQ ID NO: 746, SEQ ID NO: 747, SEQ ID NO: 748, SEQ ID NO: 749, SEQID NO: 750, SEQ ID NO: 751, SEQ ID NO: 752 or SEQ ID NO: 753, whereinthe PtIP-83 polypeptide has insecticidal activity.

In some embodiments the non-genomic polynucleotide is not the nucleicacid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 717, SEQ IDNO: 738, SEQ ID NO: 739, SEQ ID NO: 740, SEQ ID NO: 741, SEQ ID NO: 742,SEQ ID NO: 743, SEQ ID NO: 744, SEQ ID NO: 745, SEQ ID NO: 746, SEQ IDNO: 747, SEQ ID NO: 748, SEQ ID NO: 749, SEQ ID NO: 750, SEQ ID NO: 751,SEQ ID NO: 752 or SEQ ID NO: 753.

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprising an amino acid sequence having at least 40%, 45%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ ID NO:754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ ID NO:763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQID NO: 768 or SEQ ID NO: 769, wherein the PtIP-83 polypeptide hasinsecticidal activity.

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprising an amino acid sequence of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ IDNO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQID NO: 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO:756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID NO:765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768 or SEQ ID NO: 769,having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more aminoacid substitutions compared to the native amino acid at thecorresponding position of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716,SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ IDNO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762,SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ IDNO: 767, SEQ ID NO: 768 or SEQ ID NO: 769.

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprising an amino acid sequence of any one of SEQ ID NO:236-299, SEQ ID NO: 334-367, SEQ ID NO: 398-427, SEQ ID NO: 518-607, SEQID NO: 640-645, and SEQ ID NO: 728-737.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is any one of SEQ ID NO: 172-235, SEQ ID NO: 300-333, SEQ IDNO: 368-397, SEQ ID NO: 428-517, SEQ ID NO: 634-639, and SEQ ID NO:718-727.

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide variant of SEQ ID NO: 1, wherein the amino acid at position53 is Val, Ala, Cys or Thr; the amino acid at position 54 is Lys, Ala,Cys, Asp, Glu, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Ser or Thr; theamino acid at position 55 is Arg, Ala, Asp, Glu, Phe, Gly, His, Lys,Leu, Met, Asn, Gln, Ser, Thr, Val, Trp or Tyr; the amino acid atposition 56 is Leu, Glu, Phe, Ile, Met, Thr or Val; the amino acid atposition 57 is Tyr, Cys, Ile, Leu, Met, Thr or Val; the amino acid atposition 58 is Val, Cys, Ile or Leu; the amino acid at position 59 isPhe, Leu, Met, Val or Tyr; the amino acid at position 60 is Ala, Cys,Gly, Ser, Thr or Val; the amino acid at position 61 is Asp, Glu, His orSer; the amino acid at position 62 is Val, Ala, Cys, Ile, Leu or Thr;the amino acid at position 63 is Val, Ala, Cys, Ile, Leu, Met or Thr;the amino acid at position 64 is Glu, Ala, Cys, Phe, Gly, His, Ile, Leu,Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr; the amino acid atposition 65 is Leu, Ala, Cys, Phe, His, Ile, Met, Asn, Gln, Thr, Val orTrp; the amino acid at position 66 is Pro, Asp, Gly, Met, Gln or Arg;the amino acid at position 363 is Gln, Ala, Cys, Glu, Phe, Gly, His,Lys, Leu, Asn, Arg, Ser, Thr, Val or Trp; the amino acid at position 364is Ile, Ala, Cys, Glu, Phe, His, Lys, Leu, Met, Asn, Gln, Ser, Thr, Val,Trp or Tyr; the amino acid at position 365 is Leu, Ala, Glu, Phe, Gly,His, Ile, Lys, Met, Asn, Arg, Val, Trp or Tyr; the amino acid atposition 366 is Gly, Ala, Cys, Phe, His, Ile, Lys, Leu, Met, Asn, Ser,Thr or Val; the amino acid at position 367 is Ser, Ala, Cys, Asp, Glu,Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val or Trp; theamino acid at position 368 is Tyr, Ala, Cys, Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp; the aminoacid at position 369 is Leu, Ala, Cys, Asp, Phe, Gly, Ile, Met, Thr orVal; the amino acid at position 370 is Leu, Ala, Cys, Asp, Glu, Phe,Gly, His, Ile, Lys, Met, Gln, Arg, Ser, Thr, Val, Trp or Tyr; the aminoacid at position 371 is Gln, Ala, Cys, Asp, Glu, Phe, Gly, Ile, Lys,Leu, Asn, Arg, Ser, Thr, Val or Trp; the amino acid at position 372 isGln, Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Asn, Arg, Ser, Val or Tyr;the amino acid at position 373 is Asn, Ala, Cys, Asp, Phe, Gly, His,Ile, Lys, Gln, Ser, Thr, Val or Trp; the amino acid at position 556 isTrp, Phe, Thr or Tyr; the amino acid at position 557 is Arg, Cys, Asp,Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr;the amino acid at position 558 is Ala, Cys, Asp, Phe, Gly, His, Ile,Lys, Leu, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr; the amino acid atposition 559 is Lys, Ala, Cys, Phe, Gly, His, Ile, Leu, Asn, Gln, Arg,Ser, Thr, Val or Tyr; the amino acid at position 560 is Cys, Ala, Phe,Gly, Ile, Met, Asn, Arg, Ser, Thr or Val; the amino acid at position 561is Lys, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Arg, Ser,Thr, Val or Tyr; the amino acid at position 562 is Asn, Cys, Asp, Glu,Gly, His, Leu, Met, Arg, Ser, Thr, Val or Tyr; the amino acid atposition 563 is Val, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Asn, Gln,Thr or Trp; the amino acid at position 564 is Ala, Cys, Gly, Met, Gln,Ser, Thr, Val, Trp or Tyr; the amino acid at position 646 is Leu, Ala,Cys, Gly, Ile, Met, Asn, Gln, Ser, Thr or Val; the amino acid atposition 647 is Leu, Asp, Gly, Met, Asn, Gln or Thr; the amino acid atposition 648 is Met, Ala, Cys, Asp, Glu, Phe, Gly, His, Lys, Leu, Asn,Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr; the amino acid at position 649is Pro, Ala, Cys, Asp, Glu, Phe, Gly, His, Lys, Met, Asn, Gln, Arg, Ser,Thr, Trp or Tyr; the amino acid at position 650 is Thr, Ala, Cys, Asp,Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Val or Tyr; theamino acid at position 651 is Glu, Ala, Cys, Asp, Gly, His, Ile, Leu,Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Tyr; the amino acid atposition 652 is Leu, Cys, Phe, Ile, Lys, Met, Pro, Arg, Ser, Thr or Val;the amino acid at position 653 is Thr, Cys, Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Pro, Arg, Ser, Val or Trp; the amino acid at position 654is Thr, Ala, Cys, Phe, Ile, Lys, Leu, Met, Pro, Arg, Ser, Val, Trp orTyr; the amino acid at position 655 is Trp, Phe or Tyr; the amino acidat position 771 is Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu,Asn, Ser, Thr, Val, Trp or Tyr; the amino acid at position 772 is Arg,Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Ser,Thr, Val, Trp or Tyr; the amino acid at position 773 is Asp, Ala, Glu,Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp orTyr; the amino acid at position 774 is Gln, Ala, Asp, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr; the amino acidat position 775 is Val, Ala, Cys, Asp, Glu, Gly, His, Ile, Asn, Pro,Gln, Arg, Ser, Thr or Tyr; the amino acid at position 776 is Leu, Ala,Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Asn, Pro, Gln, Arg, Ser, Thr,Val or Tyr; the amino acid at position 777 is Pro, Ala, Cys, Asp, Glu,Phe, Gly, His, Lys, Leu, Met, Asn, Gln, Ser, Thr, Val, Trp or Tyr; theamino acid at position 778 is Phe, Ala, His, Ile, Leu, Met, Asn, Gln,Ser, Val, Trp or Tyr; the amino acid at position 779 is Gln, Ala, Cys,Asp, Glu, Gly, His, Lys, Leu, Asn, Pro, Arg, Ser, Thr or Val; the aminoacid at position 780 is Ala, Cys, Asn, Pro, Gln or Ser; the amino acidat position 781 is Ala, Cys, Asp, Glu, Phe, Gly, His, lie, Asn, Gln,Arg, Ser, Thr, Val, Trp or Tyr; the amino acid at position 782 is Ala,Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Pro, Gln, Arg, Ser, Thr,Val, Trp or Tyr; the amino acid at position 783 is Pro, Ala, Cys, Asp,Glu, Gly, His, Asn, Gln, Arg, Ser, Thr or Val; the amino acid atposition 784 is Leu, Ala, Glu, Phe, His, Ile, Lys, Met, Asn, Pro, Gln,Ser, Thr, Val or Trp; the amino acid at position 785 is Asn, Ala, Cys,Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Gln, Arg, Ser, Thr, Val, Trp orTyr; and the amino acid at position 786 is Tyr, Phe, Ile, Leu or Trp.

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide variant of SEQ ID NO: 1, wherein the amino acid at position1 is Met or deleted; the amino acid at position 2 is Ala or deleted; theamino acid at position 3 is Leu, Val or deleted; the amino acid atposition 4 is Val, Met or Leu; the amino acid at position 7 is Gly orSer; the amino acid at position 8 is Lys or Thr; the amino acid atposition 10 is Phe or Tyr; the amino acid at position 11 is Glu or Arg;the amino acid at position 18 is Met or Ile; the amino acid at position19 is Gly, Pro or Ala; the amino acid at position 20 is Val or deleted;the amino acid at position 21 is Leu or Val; the amino acid at position23 is Arg or Gln; the amino acid at position 37 is Val or Leu; the aminoacid at position 38 is Arg or Asn; the amino acid at position 40 is Alaor Ser; the amino acid at position 43 is Asn or Asp; the amino acid atposition 45 is Gly or Ala; the amino acid at position 46 is Gln or Glu;the amino acid at position 48 is Glu, Pro or Val; the amino acid atposition 51 is Glu or Gly; the amino acid at position 52 is Lys, Arg orThr; the amino acid at position 56 is Leu or Val; the amino acid atposition 59 is Phe or Leu; the amino acid at position 66 is Pro or Ala;the amino acid at position 67 is Val, Pro or Thr; the amino acid atposition 68 is Val, Arg, Phe or Gly; the amino acid at position 69 isGlu, Ala or Lys; the amino acid at position 70 is Trp, Thr, His, Tyr orArg; the amino acid at position 71 is Arg, Pro or deleted; the aminoacid at position 72 is Trp, Asp, Leu or deleted; the amino acid atposition 73 is Pro, Gln, Asn, His or deleted; the amino acid at position74 is Pro, Met or Thr; the amino acid at position 75 is Gln, His or Arg;the amino acid at position 76 is Ile, Met or Leu; the amino acid atposition 84 is Ile or Val; the amino acid at position 91 is Trp or Phe;the amino acid at position 93 is Thr or Ile; the amino acid at position94 is Asp or Gly; the amino acid at position 96 is Arg or Ser; the aminoacid at position 97 is Gln, Phe or Arg; the amino acid at position 98 isSer or deleted; the amino acid at position 99 is Asp or Ala; the aminoacid at position 100 is Thr or Ala; the amino acid at position 101 isGlu, Thr or Trp the amino acid at position 103 is His, Arg, Glu or Gin;the amino acid at position 105 is Thr or Pro; the amino acid at position108 is Lys, Gln or Glu; the amino acid at position 109 is Leu or Val;the amino acid at position 111 is Ala or Thr; the amino acid at position112 is Ile, Arg, Thr or deleted; the amino acid at position 113 is Gln,Ala, Gly or deleted; the amino acid at position 114 is Arg, Glu or Ile;the amino acid at position 115 is Glu or Gln; the amino acid at position116 is Glu, Asn, Gln or Arg; the amino acid at position 117 is Asn, Val,Tyr or Phe; the amino acid at position 118 is Arg or Lys; the amino acidat position 119 is Trp or Ser; the amino acid at position 122 is Thr,Lys or Ala; the amino acid at position 124 is Ala or Thr; the amino acidat position 126 is Gly or Asp; the amino acid at position 127 is Met orAla; the amino acid at position 128 is Asn or Lys; the amino acid atposition 131 is Val, lie or Thr; the amino acid at position 133 is lieor Val; the amino acid at position 134 is His or Tyr; the amino acid atposition 135 is Ala or Gly; the amino acid at position 137 is Glu orLys; the amino acid at position 139 is Gln or Glu; the amino acid atposition 140 is Val, Arg or Leu; the amino acid at position 141 is Glyor Ser; the amino acid at position 142 is Val or Pro; the amino acid atposition 144 is Thr, Leu, Phe or Tyr; the amino acid at position 145 isMet, Pro or Asn; the amino acid at position 146 is Ser, Gly or Asn; theamino acid at position 147 is Trp or Asn; the amino acid at position 148is Ser, Ala or Pro; the amino acid at position 149 is Ser or deleted;the amino acid at position 150 is Val, lie or Tyr; the amino acid atposition 152 is Arg, Ala, Val or Gly; the amino acid at position 154 isSer, Trp or Glu; the amino acid at position 156 is Leu, Asp or Gln; theamino acid at position 158 is Ser or Cys; the amino acid at position 159is Val, Thr or Ile; the amino acid at position 162 is Ser or Ala; theamino acid at position 163 is Gly or deleted; the amino acid at position164 is Phe or deleted; the amino acid at position 165 is Arg or Ala; theamino acid at position 166 is Ala, Arg, Met or Phe; the amino acid atposition 167 is Val or His; the amino acid at position 168 is Ser orAsn; the amino acid at position 169 is Val, His or Thr; the amino acidat position 170 is Phe or Val; the amino acid at position 171 is Glu,Asn or Asp; the amino acid at position 172 is Val, Ala, Arg or Glu; theamino acid at position 175 is Ser, Arg or Trp; the amino acid atposition 176 is Val or Ile; the amino acid at position 177 is Arg orIle; the amino acid at position 179 is Thr, Ile, Val or Ser; the aminoacid at position 180 is Leu, Phe or Thr; the amino acid at position 181is Gly, Thr, Gln or Ser; the amino acid at position 182 is Ala, Leu, Pheor Ile; the amino acid at position 183 is Thr or Gly; the amino acid atposition 184 is Leu, Thr, Ser or Arg; the amino acid at position 185 isArg, Gly, Asp or Ala; the amino acid at position 186 is Pro, Val or Gln;the amino acid at position 187 is Asp, Thr or Ser; the amino acid atposition 188 is His, Gly or Ala; the amino acid at position 189 is Ala,Arg, Pro or deleted; the amino acid at position 190 is Leu, Asn ordeleted; the amino acid at position 191 is Tyr or deleted; the aminoacid at position 192 is Ser, Ile, Val or Asn; the amino acid at position193 is Thr or Asp; the amino acid at position 194 is Thr or Ser; theamino acid at position 195 is Met or Thr; the amino acid at position 196is Gln, His, Leu or Ser; the amino acid at position 197 is Ala, Gly orLeu; the amino acid at position 198 is Thr, Glu or Ala; the amino acidat position 199 is Pro or Arg; the amino acid at position 200 is Asn,Ser, Thr or Gly; the amino acid at position 201 is Ala, Leu, Glu or Trp;the amino acid at position 202 is Ser, Asp, Phe or Leu; the amino acidat position 203 is His, Pro, Gly or Ser; the amino acid at position 204is Ile, Trp, His or Gly; the amino acid at position 205 is Ser, Asn orIle; the amino acid at position 206 is Ala, Gly, Asp, Tyr or Arg; theamino acid at position 207 is Phe, Val or Leu; the amino acid atposition 208 is Asn, Ser, Pro or Leu; the amino acid at position 210 isArg, Asp, Glu or Tyr; the amino acid at position 211 is lie, Ser or Thr;the amino acid at position 212 is Val, Ala or Asp; the amino acid atposition 214 is Pro or Arg; the amino acid at position 215 is Ser orThr; the amino acid at position 217 is Tyr or Phe; the amino acid atposition 218 is Arg or Ser; the amino acid at position 219 is Val orAla; the amino acid at position 220 is Cys, Leu or Ser; the amino acidat position 221 is Pro or His; the amino acid at position 222 is Leu,Arg or Ser; the amino acid at position 224 is Asn or Ser; the amino acidat position 225 is Asp, Arg or Thr; the amino acid at position 226 isThr or Asn; the amino acid at position 227 is Asp, Leu or deleted; theamino acid at position 228 is Thr or deleted; the amino acid at position229 is Tyr or deleted; the amino acid at position 230 is Leu or deleted;the amino acid at position 231 is Gly or deleted; the amino acid atposition 232 is lie or deleted; the amino acid at position 233 is Pro ordeleted; the amino acid at position 234 is Ala, Pro or deleted; theamino acid at position 235 is Asp, lie or Val; the amino acid atposition 236 is Val, Ser or Glu; the amino acid at position 237 is Ala,Phe or Tyr; the amino acid at position 238 is Ala or Thr; the amino acidat position 239 is Val, Ser or Gly; the amino acid at position 240 isLeu or Ile; the amino acid at position 243 is Asp or Glu; the amino acidat position 249 is Asn or Ser; the amino acid at position 252 is Leu orMet; the amino acid at position 257 is Thr or Ser; the amino acid atposition 259 is His or Leu; the amino acid at position 266 is Ala orVal; the amino acid at position 267 is Cys or Gly; the amino acid atposition 268 is His, Arg or Tyr; the amino acid at position 272 is Aspor Glu; the amino acid at position 273 is Val, Met, lie or Leu; theamino acid at position 274 is Val or Met; the amino acid at position 278is Gly or Ala; the amino acid at position 279 is Glu or Val; the aminoacid at position 281 is Leu or Ala; the amino acid at position 282 isAsn, Leu or Ile; the amino acid at position 285 is Asn or Ser; the aminoacid at position 286 is Lys, Asp or Glu; the amino acid at position 287is Leu or Val; the amino acid at position 290 is Pro, Gln or Arg; theamino acid at position 291 is Leu or Val; the amino acid at position 292is Lys or Val; the amino acid at position 293 is Glu or Gln; the aminoacid at position 294 is Ser, Asn or Lys; the amino acid at position 295is Thr or Ser; the amino acid at position 296 is Gln or His; the aminoacid at position 297 is Leu or Met; the amino acid at position 300 isSer or Thr; the amino acid at position 301 is Glu or Ala; the amino acidat position 302 is Ser, Pro or Ala; the amino acid at position 304 isLys or Asn; the amino acid at position 313 is Val or Ile; the amino acidat position 314 is His, Glu or Gln; the amino acid at position 315 isAla, Cys or Ser; the amino acid at position 316 is Ala or Val; the aminoacid at position 317 is Met or Ile; the amino acid at position 319 isMet or lie; the amino acid at position 320 is Val or Gly; the amino acidat position 321 is Arg or Pro; the amino acid at position 322 is lie orPhe; the amino acid at position 323 is Gly or Val; the amino acid atposition 324 is Leu or Ser; the amino acid at position 336 is Ser orAsn; the amino acid at position 339 is Asn, Lys or Arg; the amino acidat position 350 is Arg or Gln; the amino acid at position 351 is Glu orAsp; the amino acid at position 353 is Lys or Arg; the amino acid atposition 354 is Gln or Arg; the amino acid at position 355 is Phe orLeu; the amino acid at position 356 is Lys or Arg; the amino acid atposition 360 is Ile, Val or Ala; the amino acid at position 365 is Leuor Phe; the amino acid at position 371 is or Glu; the amino acid atposition 372 is or Lys; the amino acid at position 374 is Arg or Lys;the amino acid at position 376 is Phe or Leu; the amino acid at position378 is Glu or Asp; the amino acid at position 381 is Leu or Val; theamino acid at position 388 is Ala or Ser; the amino acid at position 395is Arg or Lys; the amino acid at position 396 is Glu, Gln or Gly; theamino acid at position 399 is Asp or Asn; the amino acid at position 400is Asn, Thr or Asp; the amino acid at position 401 is Thr or Ala; theamino acid at position 402 is Phe, lie or Leu; the amino acid atposition 406 is Asp or Glu; the amino acid at position 408 is Leu orMet; the amino acid at position 410 is Gly or Leu; the amino acid atposition 414 is Ala or Glu; the amino acid at position 416 is Ser, Asnor Asp; the amino acid at position 417 is Ser, Arg or Gly; the aminoacid at position 423 is Lys or Gln; the amino acid at position 431 isArg or Lys; the amino acid at position 432 is Gln or Glu; the amino acidat position 436 is Arg or Glu; the amino acid at position 440 is Asn orArg; the amino acid at position 442 is Leu or Val; the amino acid atposition 447 is Ser, Lys or Arg; the amino acid at position 448 is Alaor Ser; the amino acid at position 451 is Gln or Met; the amino acid atposition 453 is Gly or Ala; the amino acid at position 455 is Ala orVal; the amino acid at position 457 is Leu or Val; the amino acid atposition 467 is Val or Ala; the amino acid at position 471 is Gly orAla; the amino acid at position 475 is Ser or Asn; the amino acid atposition 483 is Gly or Ala; the amino acid at position 493 is Gln orGly; the amino acid at position 504 is Val or Ile; the amino acid atposition 506 is Asp or His; the amino acid at position 509 is Asp orAsn; the amino acid at position 510 is Ser or Ala; the amino acid atposition 512 is Glu or Asp; the amino acid at position 515 is Gly orSer; the amino acid at position 516 is Gln or His; the amino acid atposition 517 is lie or Leu; the amino acid at position 519 is Asp, Glyor Gln; the amino acid at position 522 is Val, Glu, Pro or Val; theamino acid at position 525 is Glu or Asp; the amino acid at position 526is Leu or Met; the amino acid at position 539 is Val or Ile; the aminoacid at position 555 is Val or Ala; the amino acid at position 557 isArg or Lys; the amino acid at position 563 is Val or Met; the amino acidat position 571 is Ser or Cys; the amino acid at position 575 is Val orGlu; the amino acid at position 577 is Met or Ile; the amino acid atposition 579 is Glu or Gln; the amino acid at position 583 is Asp orGlu; the amino acid at position 589 is Met or Leu; the amino acid atposition 590 is Met or Leu; the amino acid at position 593 is Met orIle; the amino acid at position 595 is Arg or Gln; the amino acid atposition 596 is Ser or Thr; the amino acid at position 597 is Gln orHis; the amino acid at position 607 is Ala or Val; the amino acid atposition 608 is Asp or Asn; the amino acid at position 612 is Tyr, Hisor Phe; the amino acid at position 617 is Thr or Ile; the amino acid atposition 618 is Gin or His; the amino acid at position 625 is Arg orSer; the amino acid at position 626 is Met or Ile; the amino acid atposition 628 is Leu or Ile; the amino acid at position 633 is lie orMet; the amino acid at position 634 is Leu or Met; the amino acid atposition 642 is Arg or Met; the amino acid at position 648 is Met orThr; the amino acid at position 651 is Glu or Gln; the amino acid atposition 654 is Thr, Val or Ala; the amino acid at position 658 is Glyor Arg; the amino acid at position 663 is Gly or Ala; the amino acid atposition 664 is Asp or Asn; the amino acid at position 668 is Ala orThr; the amino acid at position 669 is Gln or His; the amino acid atposition 671 is Asn or Ser the amino acid at position 675 is Ile, Val orSer; the amino acid at position 678 is Met, Ile, Ala or Thr; the aminoacid at position 682 is Pro or Gln; the amino acid at position 683 isSer or Pro; the amino acid at position 685 is Asp or Asn; the amino acidat position 694 is Asp or Gly; the amino acid at position 697 is Asn orSer; the amino acid at position 704 is Glu or Gly; the amino acid atposition 714 is Ala or Gly; the amino acid at position 721 is Ser orPhe; the amino acid at position 722 is Ser or Asn; the amino acid atposition 724 is Ser or Thr; the amino acid at position 734 is His orGln; the amino acid at position 736 is Val or Ala; the amino acid atposition 737 is Lys or Gln; the amino acid at position 739 is Ala orSer; the amino acid at position 740 is Ser or Met; the amino acid atposition 741 is Gly or Asn; the amino acid at position 742 is lie orGly; the amino acid at position 743 is Gly or deleted; the amino acid atposition 745 is Gly or Asp; the amino acid at position 751 is Thr, Seror Ala; the amino acid at position 753 is Gln or Arg; the amino acid atposition 754 is Thr or Ser; the amino acid at position 756 is Thr orIle; the amino acid at position 757 is Val or Ile; the amino acid atposition 766 is lie or Val; the amino acid at position 773 is Asp orGlu; the amino acid at position 774 is Gln or Glu; the amino acid atposition 776 is Leu or Met; the amino acid at position 777 is Pro orThr; the amino acid at position 782 is Ala, Asp or Val; the amino acidat position 786 is Tyr or Phe; the amino acid at position 787 is His orGln; the amino acid at position 788 is Tyr or Met; the amino acid atposition 789 is Ala or Arg; the amino acid at position 790 is Tyr orThr; the amino acid at position 791 is Arg or Ala; the amino acid atposition 792 is Leu or Ser; the amino acid at position 796 is Asp orGlu; the amino acid at position 797 is Ser, Thr or Ala the amino acid atposition 802 is Glu or Gln; the amino acid at position 806 is Gln, Asp,Glu or His; the amino acid at position 810 is Lys or Thr; the amino acidat position 819 is Arg or His; the amino acid at position 829 is Lys,Ser, Ala or Pro; the amino acid at position 832 is Ala, Lys or Glu; theamino acid at position 833 is Gly or Glu; the amino acid at position 842is Leu or Pro; the amino acid at position 847 is Gln or Glu; the aminoacid at position 848 is Ile or Val; the amino acid at position 849 isVal or Ala; the amino acid at position 855 is Thr or Met; the amino acidat position 860 is Ile or Val; and the amino acid at position 864 is Hisor Gln.

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide variant of SEQ ID NO: 1, wherein the amino acid at position1 is Met or deleted; the amino acid at position 2 is Ala or deleted; theamino acid at position 3 is Leu, Val, Ile or deleted; the amino acid atposition 4 is Val, Met, Ile or Leu; the amino acid at position 7 is Gly,Thr or Ser; the amino acid at position 8 is Lys, Arg, Ser or Thr; theamino acid at position 10 is Phe, Trp or Tyr; the amino acid at position11 is Glu, Asp, Lys or Arg; the amino acid at position 18 is Met, Val,Leu or Ile; the amino acid at position 19 is Gly, Pro or Ala; the aminoacid at position 20 is Val, Ile, Leu or deleted; the amino acid atposition 21 is Leu, Ile or Val; the amino acid at position 23 is Arg,Lys, Asn or Gln; the amino acid at position 37 is Val, Ile or Leu; theamino acid at position 38 is Arg, Lys, Gln or Asn; the amino acid atposition 40 is Ala, Gly, Thr or Ser; the amino acid at position 43 isAsn, Gln, Glu or Asp; the amino acid at position 45 is Gly or Ala; theamino acid at position 46 is Gln, Asp, Asn or Glu; the amino acid atposition 48 is Glu, Asp, Pro, Ile, Leu or Val; the amino acid atposition 51 is Glu, Asp, Ala or Gly; the amino acid at position 52 isLys, Arg, Ser or Thr; the amino acid at position 56 is Leu, Ile or Val;the amino acid at position 59 is Phe, Ile, Val or Leu; the amino acid atposition 66 is Pro, Gly or Ala; the amino acid at position 67 is Val,Pro, Ile, Leu, Ser or Thr; the amino acid at position 68 is Val, Arg,Phe, Ile, Leu, Lys or Gly; the amino acid at position 69 is Glu, Ala,Asp, Gly, Arg or Lys; the amino acid at position 70 is Trp, Thr, His,Tyr, Lys or Arg; the amino acid at position 71 is Arg, Pro, Lys ordeleted; the amino acid at position 72 is Trp, Asp, Leu, Ile, Val, Gluor deleted; the amino acid at position 73 is Pro, Gln, Asn, His ordeleted; the amino acid at position 74 is Pro, Met, Ser or Thr; theamino acid at position 75 is Gln, His, Asn, Lys or Arg; the amino acidat position 76 is Ile, Met, Val or Leu; the amino acid at position 84 isIle, Leu or Val; the amino acid at position 91 is Trp or Phe; the aminoacid at position 93 is Thr, Ser, Leu, Val or Ile; the amino acid atposition 94 is Asp, Glu, Ala or Gly; the amino acid at position 96 isArg, Lys, Thr or Ser; the amino acid at position 97 is Gln, Phe, Asn,Lys or Arg; the amino acid at position 98 is Ser, Thr or deleted; theamino acid at position 99 is Asp, Glu, Gly or Ala; the amino acid atposition 100 is Thr, Ser, Gly or Ala; the amino acid at position 101 isGlu, Thr, Asp, Ser or Trp the amino acid at position 103 is His, Arg,Lys, Glu or Gln; the amino acid at position 105 is Thr, Ser or Pro; theamino acid at position 108 is Lys, Arg, Asn, Asp, Gln or Glu; the aminoacid at position 109 is Leu, lie or Val; the amino acid at position 111is Ala, Ser or Thr; the amino acid at position 112 is Ile, Arg, Thr,Leu, Val, Lys, Ser or deleted; the amino acid at position 113 is Gln,Ala, Gly, Asn or deleted; the amino acid at position 114 is Arg, Glu,Lys, Asp or Ile; the amino acid at position 115 is Glu, Asp, Asn or Gln;the amino acid at position 116 is Glu, Asn, Gln, Asp, Lys or Arg; theamino acid at position 117 is Asn, Val, Tyr, Ile, Leu, Gin, Trp or Phe;the amino acid at position 118 is Arg or Lys; the amino acid at position119 is Trp, Thr or Ser; the amino acid at position 122 is Thr, Lys, Ser,Arg or Ala; the amino acid at position 124 is Ala, Gly, Ser or Thr; theamino acid at position 126 is Gly, Ala, Glu or Asp; the amino acid atposition 127 is Met, Gly or Ala; the amino acid at position 128 is Asn,Gln, Arg or Lys; the amino acid at position 131 is Val, Ile, Leu, Ser orThr; the amino acid at position 133 is Ile, Leu or Val; the amino acidat position 134 is His or Tyr; the amino acid at position 135 is Ala orGly; the amino acid at position 137 is Glu, Asp, Arg or Lys; the aminoacid at position 139 is Gln, Asn, Asp or Glu; the amino acid at position140 is Val, Arg, Ile, Lys or Leu; the amino acid at position 141 is Gly,Ala, Thr or Ser; the amino acid at position 142 is Val, Ile, Leu or Pro;the amino acid at position 144 is Thr, Leu, Phe, Ile, Val or Tyr; theamino acid at position 145 is Met, Pro, Gln or Asn; the amino acid atposition 146 is Ser, Gly, Thr, Ala, Gln or Asn; the amino acid atposition 147 is Trp, Gln, Tyr or Asn; the amino acid at position 148 isSer, Ala, Thr, Gly or Pro; the amino acid at position 149 is Ser, Thr ordeleted; the amino acid at position 150 is Val, Ile, Leu or Tyr; theamino acid at position 152 is Arg, Ala, Val, Ile, Leu, Lys or Gly; theamino acid at position 154 is Ser, Trp, Thr, Asp or Glu; the amino acidat position 156 is Leu, Asp, Ile, Val, Asn, Glu or Gln; the amino acidat position 158 is Ser, Thr or Cys; the amino acid at position 159 isVal, Thr, Leu or Ile; the amino acid at position 162 is Ser, Thr, Gly orAla; the amino acid at position 163 is Gly, Ala or deleted; the aminoacid at position 164 is Phe or deleted; the amino acid at position 165is Arg, Lys, Gly or Ala; the amino acid at position 166 is Ala, Arg,Met, Lys or Phe; the amino acid at position 167 is Val, Ile, Leu or His;the amino acid at position 168 is Ser, Thr, Gln or Asn; the amino acidat position 169 is Val, His, Ile, Leu, Ser or Thr; the amino acid atposition 170 is Phe, Ile, Leu or Val; the amino acid at position 171 isGlu, Asn, Gln or Asp; the amino acid at position 172 is Val, Ala, Arg,Ile, Leu, Gly, Lys, Asp or Glu; the amino acid at position 175 is Ser,Arg, Thr, Lys or Trp; the amino acid at position 176 is Val, Leu or Ile;the amino acid at position 177 is Arg, Lys, Leu, Val or Ile; the aminoacid at position 179 is Thr, Ile, Val, Leu or Ser; the amino acid atposition 180 is Leu, Phe, Ile, Val, Ser or Thr; the amino acid atposition 181 is Gly, Thr, Gln, Asn or Ser; the amino acid at position182 is Ala, Leu, Phe, Val or Ile; the amino acid at position 183 is Thr,Ser, Ala or Gly; the amino acid at position 184 is Leu, Thr, Ser, Ile,Val, Lys or Arg; the amino acid at position 185 is Arg, Gly, Asp, Lys,Glu or Ala; the amino acid at position 186 is Pro, Val, Ile, Leu, Asn orGln; the amino acid at position 187 is Asp, Thr, Glu or Ser; the aminoacid at position 188 is His, Gly or Ala; the amino acid at position 189is Ala, Arg, Pro, Lys, Gly or deleted; the amino acid at position 190 isLeu, Asn, Ile, Val, Gln or deleted; the amino acid at position 191 isTyr or deleted; the amino acid at position 192 is Ser, Ile, Val, Leu,Thr or Asn; the amino acid at position 193 is Thr, Ser, Glu or Asp; theamino acid at position 194 is Thr or Ser; the amino acid at position 195is Met or Thr; the amino acid at position 196 is Gln, His, Leu, Asn,Ile, Val, Thr or Ser; the amino acid at position 197 is Ala, Gly, Ile,Val or Leu; the amino acid at position 198 is Thr, Glu, Ser, Asp, Gly orAla; the amino acid at position 199 is Pro, Lys or Arg; the amino acidat position 200 is Asn, Ser, Thr, Gln, Ala or Gly; the amino acid atposition 201 is Ala, Leu, Glu, lie, Asp or Trp; the amino acid atposition 202 is Ser, Asp, Phe, Ile, Val, Thr, Glu or Leu; the amino acidat position 203 is His, Pro, Gly, Ala, Thr or Ser; the amino acid atposition 204 is lie, Trp, His, Leu, Val, Ala or Gly; the amino acid atposition 205 is Ser, Asn, Leu, Val, Thr, Gln or Ile; the amino acid atposition 206 is Ala, Gly, Asp, Tyr, Glu, Lys or Arg; the amino acid atposition 207 is Phe, Val, lie or Leu; the amino acid at position 208 isAsn, Ser, Pro, Gln, Thr, Val, lie or Leu; the amino acid at position 210is Arg, Asp, Glu, Lys, Ser or Tyr; the amino acid at position 211 isIle, Ser, Leu, Val or Thr; the amino acid at position 212 is Val, Ala,Ile, Leu, Glu, Gly or Asp; the amino acid at position 214 is Pro, Lys orArg; the amino acid at position 215 is Ser or Thr; the amino acid atposition 217 is Tyr or Phe; the amino acid at position 218 is Arg, Lys,Thr or Ser; the amino acid at position 219 is Val, Ile, Leu or Ala; theamino acid at position 220 is Cys, Leu, Ile, Val, Thr or Ser; the aminoacid at position 221 is Pro or His; the amino acid at position 222 isLeu, Arg, Lys, Ile, Val, Thr or Ser; the amino acid at position 224 isAsn, Gln, Thr or Ser; the amino acid at position 225 is Asp, Arg, Glu,Lys, Ser or Thr; the amino acid at position 226 is Thr, Ser, Gln or Asn;the amino acid at position 227 is Asp, Leu, Glu, Ile, Val or deleted;the amino acid at position 228 is Thr, Ser or deleted; the amino acid atposition 229 is Tyr or deleted; the amino acid at position 230 is Leu,Ile, Val or deleted; the amino acid at position 231 is Gly, Ala ordeleted; the amino acid at position 232 is Ile, Leu, Val or deleted; theamino acid at position 233 is Pro or deleted; the amino acid at position234 is Ala, Pro, Gly or deleted; the amino acid at position 235 is Asp,Ile, Leu, Glu or Val; the amino acid at position 236 is Val, Ser, Ile,Leu, Thr, Asp or Glu; the amino acid at position 237 is Ala, Phe or Tyr;the amino acid at position 238 is Ala, Gly, Ser or Thr; the amino acidat position 239 is Val, Ser, Ile, Leu, Thr, Ala or Gly; the amino acidat position 240 is Leu, Val or Ile; the amino acid at position 243 isAsp or Glu; the amino acid at position 249 is Asn, Gln, Thr or Ser; theamino acid at position 252 is Leu, Ile, Val or Met; the amino acid atposition 257 is Thr or Ser; the amino acid at position 259 is His, Ile,Val or Leu; the amino acid at position 266 is Ala, lie, Leu or Val; theamino acid at position 267 is Cys, Ala or Gly; the amino acid atposition 268 is His, Arg, Lys or Tyr; the amino acid at position 272 isAsp or Glu; the amino acid at position 273 is Val, Met, lie or Leu; theamino acid at position 274 is Val, Ile, Leu or Met; the amino acid atposition 278 is Gly or Ala; the amino acid at position 279 is Glu, Asp,Gly or Val; the amino acid at position 281 is Leu, Ile, Val, Gly or Ala;the amino acid at position 282 is Asn, Leu or lie; the amino acid atposition 285 is Asn, Gln, Thr or Ser; the amino acid at position 286 isLys, Asp, Arg or Glu; the amino acid at position 287 is Leu, lie or Val;the amino acid at position 290 is Pro, Gln, Asn, Lys or Arg; the aminoacid at position 291 is Leu, lie or Val; the amino acid at position 292is Lys, Arg, Ile, Leu or Val; the amino acid at position 293 is Glu,Asp, Asn or Gln; the amino acid at position 294 is Ser, Asn, Thr, Gln,Arg or Lys; the amino acid at position 295 is Thr or Ser; the amino acidat position 296 is Gln, Asn or His; the amino acid at position 297 isLeu, Ile, Val or Met; the amino acid at position 300 is Ser or Thr; theamino acid at position 301 is Glu, Asp, Gly or Ala; the amino acid atposition 302 is Ser, Pro, Thr, Gly or Ala; the amino acid at position304 is Lys, Arg, Gln or Asn; the amino acid at position 313 is Val, Leuor Ile; the amino acid at position 314 is His, Glu, Asn, Asp or Gln; theamino acid at position 315 is Ala, Cys, Gly, Thr or Ser; the amino acidat position 316 is Ala, Ile, Leu or Val; the amino acid at position 317is Met, Leu, Val or Ile; the amino acid at position 319 is Met, Leu, Valor lie; the amino acid at position 320 is Val, Ile, Leu, Ala or Gly; theamino acid at position 321 is Arg, Lys or Pro; the amino acid atposition 322 is Ile, Leu, Val or Phe; the amino acid at position 323 isGly, Ile, Leu or Val; the amino acid at position 324 is Leu, Ile, Val,Thr or Ser; the amino acid at position 336 is Ser, Thr, Gln or Asn; theamino acid at position 339 is Asn, Lys, Gln or Arg; the amino acid atposition 350 is Arg, Lys, Asn or Gln; the amino acid at position 351 isGlu or Asp; the amino acid at position 353 is Lys or Arg; the amino acidat position 354 is Gln, Asn, Lys or Arg; the amino acid at position 355is Phe, Ile, Leu or Leu; the amino acid at position 356 is Lys or Arg;the amino acid at position 360 is Ile, Val, Leu, Gly or Ala; the aminoacid at position 365 is Leu, Ile, Val or Phe; the amino acid at position371 is or Glu or Asp; the amino acid at position 372 is or Lys or Arg;the amino acid at position 374 is Arg or Lys; the amino acid at position376 is Phe, Ile, Val or Leu; the amino acid at position 378 is Glu orAsp; the amino acid at position 381 is Leu, lie or Val; the amino acidat position 388 is Ala, Thr, Gly or Ser; the amino acid at position 395is Arg or Lys; the amino acid at position 396 is Glu, Gln, Asp, Asn, Alaor Gly; the amino acid at position 399 is Asp, Gln, Glu or Asn; theamino acid at position 400 is Asn, Thr, Ser, Glu, Gln or Asp; the aminoacid at position 401 is Thr, Ser, Gly or Ala; the amino acid at position402 is Phe, Ile, Val or Leu; the amino acid at position 406 is Asp orGlu; the amino acid at position 408 is Leu, Ile, Val or Met; the aminoacid at position 410 is Gly, Ile, Val, Ala or Leu; the amino acid atposition 414 is Ala, Gly, Asp or Glu; the amino acid at position 416 isSer, Asn, Thr, Gln, Glu or Asp; the amino acid at position 417 is Ser,Arg, Lys, Thr, Ala or Gly; the amino acid at position 423 is Lys, Arg,Asn or Gln; the amino acid at position 431 is Arg or Lys; the amino acidat position 432 is Gln, Asn, Asp or Glu; the amino acid at position 436is Arg, Lys, Asp or Glu; the amino acid at position 440 is Asn, Gln, Lysor Arg; the amino acid at position 442 is Leu, lie or Val; the aminoacid at position 447 is Ser, Lys, Thr or Arg; the amino acid at position448 is Ala, Gly, Thr or Ser; the amino acid at position 451 is Gln, Asnor Met; the amino acid at position 453 is Gly or Ala; the amino acid atposition 455 is Ala, Leu, lie or Val; the amino acid at position 457 isLeu, lie or Val; the amino acid at position 467 is Val, Ile, Leu, Gly orAla; the amino acid at position 471 is Gly or Ala; the amino acid atposition 475 is Ser, Thr, Gln or Asn; the amino acid at position 483 isGly or Ala; the amino acid at position 493 is Gln, Asn or Gly; the aminoacid at position 504 is Val, Leu or Ile; the amino acid at position 506is Asp, Glu or His; the amino acid at position 509 is Asp, Glu, Gln orAsn; the amino acid at position 510 is Ser, Thr, Gly or Ala; the aminoacid at position 512 is Glu or Asp; the amino acid at position 515 isGly, Ala, Thr or Ser; the amino acid at position 516 is Gln, Asn or His;the amino acid at position 517 is Ile, Val or Leu; the amino acid atposition 519 is Asp, Asn, Glu, Gly or Gln; the amino acid at position522 is Val, Glu, Pro, Ile, Leu or Asp; the amino acid at position 525 isGlu or Asp; the amino acid at position 526 is Leu, lie, Val or Met; theamino acid at position 539 is Val, Leu or Ile; the amino acid atposition 555 is Val, Leu, lie or Ala; the amino acid at position 557 isArg or Lys; the amino acid at position 563 is Val, Leu, lie or Met; theamino acid at position 571 is Ser, Thr or Cys; the amino acid atposition 575 is Val, Leu, Ile, Asp or Glu; the amino acid at position577 is Met, Leu, Val or Ile; the amino acid at position 579 is Glu, Asp,Asn or Gln; the amino acid at position 583 is Asp or Glu; the amino acidat position 589 is Met, Ile, Val or Leu; the amino acid at position 590is Met, lie, Val or Leu; the amino acid at position 593 is Met, Leu, Valor Ile; the amino acid at position 595 is Arg, Lys, Asn or Gln; theamino acid at position 596 is Ser or Thr; the amino acid at position 597is Gln, Asn or His; the amino acid at position 607 is Ala, Gly, Ile, Leuor Val; the amino acid at position 608 is Asp, Glu, Gln or Asn; theamino acid at position 612 is Tyr, His or Phe; the amino acid atposition 617 is Thr, Ser, Leu, Val or Ile; the amino acid at position618 is Gln, Asn or His; the amino acid at position 625 is Arg, Lys, Thror Ser; the amino acid at position 626 is Met, Leu, Val or Ile; theamino acid at position 628 is Leu, Val or Ile; the amino acid atposition 633 is Ile, Leu, Val or Met; the amino acid at position 634 isLeu, Ile, Val or Met; the amino acid at position 642 is Arg, Lys or Met;the amino acid at position 648 is Met, Ser or Thr; the amino acid atposition 651 is Glu, Asp, Asn or Gln; the amino acid at position 654 isThr, Val, Ser, Ile, Leu, Gly or Ala; the amino acid at position 658 isGly, Lys, Ala or Arg; the amino acid at position 663 is Gly or Ala; theamino acid at position 664 is Asp, Glu, Gln or Asn; the amino acid atposition 668 is Ala, Gly, Ser or Thr; the amino acid at position 669 isGln, Asn or His; the amino acid at position 671 is Asn, Gln, Thr or Serthe amino acid at position 675 is Ile, Val, Ile, Thr or Ser; the aminoacid at position 678 is Met, Ile, Ala, Leu, Ser or Thr; the amino acidat position 682 is Pro, Asn or Gln; the amino acid at position 683 isSer, Thr or Pro; the amino acid at position 685 is Asp, Glu, Asp or Asn;the amino acid at position 694 is Asp, Glu, Ala or Gly; the amino acidat position 697 is Asn, Gln, Thr or Ser; the amino acid at position 704is Glu, Asp, Ala or Gly; the amino acid at position 714 is Ala or Gly;the amino acid at position 721 is Ser, Thr or Phe; the amino acid atposition 722 is Ser, Thr, Gln or Asn; the amino acid at position 724 isSer or Thr; the amino acid at position 734 is His, Asn or Gln; the aminoacid at position 736 is Val, Leu, lie or Ala; the amino acid at position737 is Lys, Arg, Asn or Gln; the amino acid at position 739 is Ala, Gly,Thr or Ser; the amino acid at position 740 is Ser, Thr or Met; the aminoacid at position 741 is Gly, Ala, Gln or Asn; the amino acid at position742 is Ile, Leu, Val, Ala or Gly; the amino acid at position 743 is Glyor deleted; the amino acid at position 745 is Gly, Ala, Glu or Asp; theamino acid at position 751 is Thr, Ser, Gly or Ala; the amino acid atposition 753 is Gln, Asn, Lys or Arg; the amino acid at position 754 isThr or Ser; the amino acid at position 756 is Thr, Ser, Leu, Val or Ile;the amino acid at position 757 is Val, Leu or Ile; the amino acid atposition 766 is Ile, Leu or Val; the amino acid at position 773 is Aspor Glu; the amino acid at position 774 is Gln, Asn, Asp or Glu; theamino acid at position 776 is Leu, Ile, Val or Met; the amino acid atposition 777 is Pro, Ser or Thr; the amino acid at position 782 is Ala,Asp, Glu, Ile, Leu or Val; the amino acid at position 786 is Tyr or Phe;the amino acid at position 787 is His, Asn or Gln; the amino acid atposition 788 is Tyr or Met; the amino acid at position 789 is Ala, Lysor Arg; the amino acid at position 790 is Tyr or Thr; the amino acid atposition 791 is Arg, Lys, Gly or Ala; the amino acid at position 792 isLeu, Ile, Val, Thr or Ser; the amino acid at position 796 is Asp or Glu;the amino acid at position 797 is Ser, Thr or Ala the amino acid atposition 802 is Glu, Lys, Asp, Asn or Gln; the amino acid at position806 is Gln, Asp, Glu, Asn or His; the amino acid at position 810 is Lys,Arg or Thr; the amino acid at position 819 is Arg, Lys or His; the aminoacid at position 829 is Lys, Ser, Ala or Pro; the amino acid at position832 is Ala, Lys, Arg, Asp or Glu; the amino acid at position 833 is Gly,Ala, Asp or Glu; the amino acid at position 842 is Leu, Ile, Val or Pro;the amino acid at position 847 is Gln, Asn, Asp or Glu; the amino acidat position 848 is Ile, Leu or Val; the amino acid at position 849 isVal, Leu, Ile, Gly or Ala; the amino acid at position 855 is Thr, Ser orMet; the amino acid at position 860 is Ile, Leu or Val; the amino acidat position 864 is His, Asn or Gln;

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide variant of SEQ ID NO: 1, wherein the amino acid at position1 is Met or deleted; the amino acid at position 2 is Ala or deleted; theamino acid at position 3 is Leu, Val, Ile or deleted; the amino acid atposition 4 is Val, Met, Ile or Leu; the amino acid at position 7 is Gly,Thr or Ser; the amino acid at position 8 is Lys, Arg, Ser or Thr; theamino acid at position 10 is Phe, Trp or Tyr; the amino acid at position11 is Glu, Asp, Lys or Arg; the amino acid at position 18 is Met, Val,Leu or Ile; the amino acid at position 19 is Gly, Pro or Ala; the aminoacid at position 20 is Val, Ile, Leu or deleted; the amino acid atposition 21 is Leu, Ile or Val; the amino acid at position 23 is Arg,Lys, Asn or Gln; the amino acid at position 37 is Val, Ile or Leu; theamino acid at position 38 is Arg, Lys, Gln or Asn; the amino acid atposition 40 is Ala, Gly, Thr or Ser; the amino acid at position 43 isAsn, Gln, Glu or Asp; the amino acid at position 45 is Gly or Ala; theamino acid at position 46 is Gln, Asp, Asn or Glu; the amino acid atposition 48 is Glu, Asp, Pro, Ile, Leu or Val; the amino acid atposition 51 is Glu, Asp, Ala or Gly; the amino acid at position 52 isLys, Arg, Ser or Thr; the amino acid at position 53 is Val, Ala, Cys orThr; the amino acid at position 54 is Lys, Ala, Cys, Asp, Glu, Gly, His,Ile, Leu, Met, Asn, Gln, Arg, Ser or Thr; the amino acid at position 55is Arg, Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Gin, Ser, Thr,Val, Trp or Tyr; the amino acid at position 56 is Leu, Glu, Phe, Ile,Met, Thr or Val; the amino acid at position 57 is Tyr, Cys, Ile, Leu,Met, Thr or Val; the amino acid at position 58 is Val, Cys, Ile or Leu;the amino acid at position 59 is Phe, Leu, Met, Val or Tyr; the aminoacid at position 60 is Ala, Cys, Gly, Ser, Thr or Val; the amino acid atposition 61 is Asp, Glu, His or Ser; the amino acid at position 62 isVal, Ala, Cys, Ile, Leu or Thr; the amino acid at position 63 is Val,Ala, Cys, Ile, Leu, Met or Thr; the amino acid at position 64 is Glu,Ala, Cys, Phe, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val,Trp or Tyr; the amino acid at position 65 is Leu, Ala, Cys, Phe, His,Ile, Met, Asn, Gln, Thr, Val or Trp; the amino acid at position 66 isPro, Asp, Gly, Met, Gln or Arg; the amino acid at position 67 is Val,Pro, Ile, Leu, Ser or Thr; the amino acid at position 68 is Val, Arg,Phe, Ile, Leu, Lys or Gly; the amino acid at position 69 is Glu, Ala,Asp, Gly, Arg or Lys; the amino acid at position 70 is Trp, Thr, His,Tyr, Lys or Arg; the amino acid at position 71 is Arg, Pro, Lys ordeleted; the amino acid at position 72 is Trp, Asp, Leu, Ile, Val, Gluor deleted; the amino acid at position 73 is Pro, Gln, Asn, His ordeleted; the amino acid at position 74 is Pro, Met, Ser or Thr; theamino acid at position 75 is Gin, His, Asn, Lys or Arg; the amino acidat position 76 is Ile, Met, Val or Leu; the amino acid at position 84 isIle, Leu or Val; the amino acid at position 91 is Trp or Phe; the aminoacid at position 93 is Thr, Ser, Leu, Val or Ile; the amino acid atposition 94 is Asp, Glu, Ala or Gly; the amino acid at position 96 isArg, Lys, Thr or Ser; the amino acid at position 97 is Gln, Phe, Asn,Lys or Arg; the amino acid at position 98 is Ser, Thr or deleted; theamino acid at position 99 is Asp, Glu, Gly or Ala; the amino acid atposition 100 is Thr, Ser, Gly or Ala; the amino acid at position 101 isGlu, Thr, Asp, Ser or Trp the amino acid at position 103 is His, Arg,Lys, Glu or Gin; the amino acid at position 105 is Thr, Ser or Pro; theamino acid at position 108 is Lys, Arg, Asn, Asp, Gln or Glu; the aminoacid at position 109 is Leu, lie or Val; the amino acid at position 111is Ala, Ser or Thr; the amino acid at position 112 is Ile, Arg, Thr,Leu, Val, Lys, Ser or deleted; the amino acid at position 113 is Gln,Ala, Gly, Asn or deleted; the amino acid at position 114 is Arg, Glu,Lys, Asp or Ile; the amino acid at position 115 is Glu, Asp, Asn or Gln;the amino acid at position 116 is Glu, Asn, Gln, Asp, Lys or Arg; theamino acid at position 117 is Asn, Val, Tyr, Ile, Leu, Gln, Trp or Phe;the amino acid at position 118 is Arg or Lys; the amino acid at position119 is Trp, Thr or Ser; the amino acid at position 122 is Thr, Lys, Ser,Arg or Ala; the amino acid at position 124 is Ala, Gly, Ser or Thr; theamino acid at position 126 is Gly, Ala, Glu or Asp; the amino acid atposition 127 is Met, Gly or Ala; the amino acid at position 128 is Asn,Gln, Arg or Lys; the amino acid at position 131 is Val, Ile, Leu, Ser orThr; the amino acid at position 133 is Ile, Leu or Val; the amino acidat position 134 is His or Tyr; the amino acid at position 135 is Ala orGly; the amino acid at position 137 is Glu, Asp, Arg or Lys; the aminoacid at position 139 is Gln, Asn, Asp or Glu; the amino acid at position140 is Val, Arg, Ile, Lys or Leu; the amino acid at position 141 is Gly,Ala, Thr or Ser; the amino acid at position 142 is Val, Ile, Leu or Pro;the amino acid at position 144 is Thr, Leu, Phe, Ile, Val or Tyr; theamino acid at position 145 is Met, Pro, Gln or Asn; the amino acid atposition 146 is Ser, Gly, Thr, Ala, Gln or Asn; the amino acid atposition 147 is Trp, Gln, Tyr or Asn; the amino acid at position 148 isSer, Ala, Thr, Gly or Pro; the amino acid at position 149 is Ser, Thr ordeleted; the amino acid at position 150 is Val, Ile, Leu or Tyr; theamino acid at position 152 is Arg, Ala, Val, Ile, Leu, Lys or Gly; theamino acid at position 154 is Ser, Trp, Thr, Asp or Glu; the amino acidat position 156 is Leu, Asp, Ile, Val, Asn, Glu or Gln; the amino acidat position 158 is Ser, Thr or Cys; the amino acid at position 159 isVal, Thr, Leu or Ile; the amino acid at position 162 is Ser, Thr, Gly orAla; the amino acid at position 163 is Gly, Ala or deleted; the aminoacid at position 164 is Phe or deleted; the amino acid at position 165is Arg, Lys, Gly or Ala; the amino acid at position 166 is Ala, Arg,Met, Lys or Phe; the amino acid at position 167 is Val, Ile, Leu or His;the amino acid at position 168 is Ser, Thr, Gln or Asn; the amino acidat position 169 is Val, His, Ile, Leu, Ser or Thr; the amino acid atposition 170 is Phe, Ile, Leu or Val; the amino acid at position 171 isGlu, Asn, Gln or Asp; the amino acid at position 172 is Val, Ala, Arg,Ile, Leu, Gly, Lys, Asp or Glu; the amino acid at position 175 is Ser,Arg, Thr, Lys or Trp; the amino acid at position 176 is Val, Leu or Ile;the amino acid at position 177 is Arg, Lys, Leu, Val or Ile; the aminoacid at position 179 is Thr, Ile, Val, Leu or Ser; the amino acid atposition 180 is Leu, Phe, Ile, Val, Ser or Thr; the amino acid atposition 181 is Gly, Thr, Gln, Asn or Ser; the amino acid at position182 is Ala, Leu, Phe, Val or Ile; the amino acid at position 183 is Thr,Ser, Ala or Gly; the amino acid at position 184 is Leu, Thr, Ser, Ile,Val, Lys or Arg; the amino acid at position 185 is Arg, Gly, Asp, Lys,Glu or Ala; the amino acid at position 186 is Pro, Val, Ile, Leu, Asn orGln; the amino acid at position 187 is Asp, Thr, Glu or Ser; the aminoacid at position 188 is His, Gly or Ala; the amino acid at position 189is Ala, Arg, Pro, Lys, Gly or deleted; the amino acid at position 190 isLeu, Asn, Ile, Val, Gln or deleted; the amino acid at position 191 isTyr or deleted; the amino acid at position 192 is Ser, Ile, Val, Leu,Thr or Asn; the amino acid at position 193 is Thr, Ser, Glu or Asp; theamino acid at position 194 is Thr or Ser; the amino acid at position 195is Met or Thr; the amino acid at position 196 is Gln, His, Leu, Asn,Ile, Val, Thr or Ser; the amino acid at position 197 is Ala, Gly, Ile,Val or Leu; the amino acid at position 198 is Thr, Glu, Ser, Asp, Gly orAla; the amino acid at position 199 is Pro, Lys or Arg; the amino acidat position 200 is Asn, Ser, Thr, Gln, Ala or Gly; the amino acid atposition 201 is Ala, Leu, Glu, Ile, Asp or Trp; the amino acid atposition 202 is Ser, Asp, Phe, Ile, Val, Thr, Glu or Leu; the amino acidat position 203 is His, Pro, Gly, Ala, Thr or Ser; the amino acid atposition 204 is Ile, Trp, His, Leu, Val, Ala or Gly; the amino acid atposition 205 is Ser, Asn, Leu, Val, Thr, Gln or Ile; the amino acid atposition 206 is Ala, Gly, Asp, Tyr, Glu, Lys or Arg; the amino acid atposition 207 is Phe, Val, lie or Leu; the amino acid at position 208 isAsn, Ser, Pro, Gln, Thr, Val, lie or Leu; the amino acid at position 210is Arg, Asp, Glu, Lys, Ser or Tyr; the amino acid at position 211 isIle, Ser, Leu, Val or Thr; the amino acid at position 212 is Val, Ala,Ile, Leu, Glu, Gly or Asp; the amino acid at position 214 is Pro, Lys orArg; the amino acid at position 215 is Ser or Thr; the amino acid atposition 217 is Tyr or Phe; the amino acid at position 218 is Arg, Lys,Thr or Ser; the amino acid at position 219 is Val, Ile, Leu or Ala; theamino acid at position 220 is Cys, Leu, Ile, Val, Thr or Ser; the aminoacid at position 221 is Pro or His; the amino acid at position 222 isLeu, Arg, Lys, Ile, Val, Thr or Ser; the amino acid at position 224 isAsn, Gln, Thr or Ser; the amino acid at position 225 is Asp, Arg, Glu,Lys, Ser or Thr; the amino acid at position 226 is Thr, Ser, Gln or Asn;the amino acid at position 227 is Asp, Leu, Glu, Ile, Val or deleted;the amino acid at position 228 is Thr, Ser or deleted; the amino acid atposition 229 is Tyr or deleted; the amino acid at position 230 is Leu,Ile, Val or deleted; the amino acid at position 231 is Gly, Ala ordeleted; the amino acid at position 232 is Ile, Leu, Val or deleted; theamino acid at position 233 is Pro or deleted; the amino acid at position234 is Ala, Pro, Gly or deleted; the amino acid at position 235 is Asp,lie, Leu, Glu or Val; the amino acid at position 236 is Val, Ser, Ile,Leu, Thr, Asp or Glu; the amino acid at position 237 is Ala, Phe or Tyr;the amino acid at position 238 is Ala, Gly, Ser or Thr; the amino acidat position 239 is Val, Ser, Ile, Leu, Thr, Ala or Gly; the amino acidat position 240 is Leu, Val or Ile; the amino acid at position 243 isAsp or Glu; the amino acid at position 249 is Asn, Gln, Thr or Ser; theamino acid at position 252 is Leu, Ile, Val or Met; the amino acid atposition 257 is Thr or Ser; the amino acid at position 259 is His, Ile,Val or Leu; the amino acid at position 266 is Ala, Ile, Leu or Val; theamino acid at position 267 is Cys, Ala or Gly; the amino acid atposition 268 is His, Arg, Lys or Tyr; the amino acid at position 272 isAsp or Glu; the amino acid at position 273 is Val, Met, lie or Leu; theamino acid at position 274 is Val, Ile, Leu or Met; the amino acid atposition 278 is Gly or Ala; the amino acid at position 279 is Glu, Asp,Gly or Val; the amino acid at position 281 is Leu, Ile, Val, Gly or Ala;the amino acid at position 282 is Asn, Leu or Ile; the amino acid atposition 285 is Asn, Gln, Thr or Ser; the amino acid at position 286 isLys, Asp, Arg or Glu; the amino acid at position 287 is Leu, lie or Val;the amino acid at position 290 is Pro, Gln, Asn, Lys or Arg; the aminoacid at position 291 is Leu, lie or Val; the amino acid at position 292is Lys, Arg, Ile, Leu or Val; the amino acid at position 293 is Glu,Asp, Asn or Gln; the amino acid at position 294 is Ser, Asn, Thr, Gln,Arg or Lys; the amino acid at position 295 is Thr or Ser; the amino acidat position 296 is Gln, Asn or His; the amino acid at position 297 isLeu, Ile, Val or Met; the amino acid at position 300 is Ser or Thr; theamino acid at position 301 is Glu, Asp, Gly or Ala; the amino acid atposition 302 is Ser, Pro, Thr, Gly or Ala; the amino acid at position304 is Lys, Arg, Gln or Asn; the amino acid at position 313 is Val, Leuor Ile; the amino acid at position 314 is His, Glu, Asn, Asp or Gln; theamino acid at position 315 is Ala, Cys, Gly, Thr or Ser; the amino acidat position 316 is Ala, Ile, Leu or Val; the amino acid at position 317is Met, Leu, Val or Ile; the amino acid at position 319 is Met, Leu, Valor Ile; the amino acid at position 320 is Val, Ile, Leu, Ala or Gly; theamino acid at position 321 is Arg, Lys or Pro; the amino acid atposition 322 is Ile, Leu, Val or Phe; the amino acid at position 323 isGly, Ile, Leu or Val; the amino acid at position 324 is Leu, Ile, Val,Thr or Ser; the amino acid at position 336 is Ser, Thr, Gln or Asn; theamino acid at position 339 is Asn, Lys, Gln or Arg; the amino acid atposition 350 is Arg, Lys, Asn or Gln; the amino acid at position 351 isGlu or Asp; the amino acid at position 353 is Lys or Arg; the amino acidat position 354 is Gln, Asn, Lys or Arg; the amino acid at position 355is Phe, Ile, Leu or Leu; the amino acid at position 356 is Lys or Arg;the amino acid at position 360 is Ile, Val, Leu, Gly or Ala; the aminoacid at position 363 is Gln, Ala, Cys, Glu, Phe, Gly, His, Lys, Leu,Asn, Arg, Ser, Thr, Val or Trp; the amino acid at position 364 is lie,Ala, Cys, Glu, Phe, His, Lys, Leu, Met, Asn, Gln, Ser, Thr, Val, Trp orTyr; the amino acid at position 365 is Leu, Ala, Glu, Phe, Gly, His,Ile, Lys, Met, Asn, Arg, Val, Trp or Tyr; the amino acid at position 366is Gly, Ala, Cys, Phe, His, Ile, Lys, Leu, Met, Asn, Ser, Thr or Val;the amino acid at position 367 is Ser, Ala, Cys, Asp, Glu, Phe, Gly,His, Ile, Leu, Met, Asn, Pro, Gin, Arg, Thr, Val or Trp; the amino acidat position 368 is Tyr, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys,Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp; the amino acid atposition 369 is Leu, Ala, Cys, Asp, Phe, Gly, Ile, Met, Thr or Val; theamino acid at position 370 is Leu, Ala, Cys, Asp, Glu, Phe, Gly, His,Ile, Lys, Met, Gln, Arg, Ser, Thr, Val, Trp or Tyr; the amino acid atposition 371 is Gln, Ala, Cys, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Asn,Arg, Ser, Thr, Val or Trp; the amino acid at position 372 is Gln, Ala,Cys, Asp, Phe, Gly, His, Ile, Leu, Asn, Arg, Ser, Val or Tyr; the aminoacid at position 373 is Asn, Ala, Cys, Asp, Phe, Gly, His, Ile, Lys,Gln, Ser, Thr, Val or Trp; the amino acid at position 374 is Arg or Lys;the amino acid at position 376 is Phe, Ile, Val or Leu; the amino acidat position 378 is Glu or Asp; the amino acid at position 381 is Leu,lie or Val; the amino acid at position 388 is Ala, Thr, Gly or Ser; theamino acid at position 395 is Arg or Lys; the amino acid at position 396is Glu, Gln, Asp, Asn, Ala or Gly; the amino acid at position 399 isAsp, Gln, Glu or Asn; the amino acid at position 400 is Asn, Thr, Ser,Glu, Gln or Asp; the amino acid at position 401 is Thr, Ser, Gly or Ala;the amino acid at position 402 is Phe, Ile, Val or Leu; the amino acidat position 406 is Asp or Glu; the amino acid at position 408 is Leu,Ile, Val or Met; the amino acid at position 410 is Gly, Ile, Val, Ala orLeu; the amino acid at position 414 is Ala, Gly, Asp or Glu; the aminoacid at position 416 is Ser, Asn, Thr, Gln, Glu or Asp; the amino acidat position 417 is Ser, Arg, Lys, Thr, Ala or Gly; the amino acid atposition 423 is Lys, Arg, Asn or Gln; the amino acid at position 431 isArg or Lys; the amino acid at position 432 is Gln, Asn, Asp or Glu; theamino acid at position 436 is Arg, Lys, Asp or Glu; the amino acid atposition 440 is Asn, Gln, Lys or Arg; the amino acid at position 442 isLeu, lie or Val; the amino acid at position 447 is Ser, Lys, Thr or Arg;the amino acid at position 448 is Ala, Gly, Thr or Ser; the amino acidat position 451 is Gln, Asn or Met; the amino acid at position 453 isGly or Ala; the amino acid at position 455 is Ala, Leu, lie or Val; theamino acid at position 457 is Leu, lie or Val; the amino acid atposition 467 is Val, lie, Leu, Gly or Ala; the amino acid at position471 is Gly or Ala; the amino acid at position 475 is Ser, Thr, Gln orAsn; the amino acid at position 483 is Gly or Ala; the amino acid atposition 493 is Gln, Asn or Gly; the amino acid at position 504 is Val,Leu or Ile; the amino acid at position 506 is Asp, Glu or His; the aminoacid at position 509 is Asp, Glu, Gln or Asn; the amino acid at position510 is Ser, Thr, Gly or Ala; the amino acid at position 512 is Glu orAsp; the amino acid at position 515 is Gly, Ala, Thr or Ser; the aminoacid at position 516 is Gln, Asn or His; the amino acid at position 517is Ile, Val or Leu; the amino acid at position 519 is Asp, Asn, Glu, Glyor Gln; the amino acid at position 522 is Val, Glu, Pro, Ile, Leu orAsp; the amino acid at position 525 is Glu or Asp; the amino acid atposition 526 is Leu, Ile, Val or Met; the amino acid at position 539 isVal, Leu or Ile; the amino acid at position 555 is Val, Leu, lie or Ala;the amino acid at position 556 is Trp, Phe, Thr or Tyr; the amino acidat position 557 is Arg, Cys, Asp, Gly, His, Ile, Lys, Leu, Met, Asn,Pro, Gln, Ser, Thr, Val, Trp or Tyr; the amino acid at position 558 isAla, Cys, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser,Val, Trp or Tyr; the amino acid at position 559 is Lys, Ala, Cys, Phe,Gly, His, Ile, Leu, Asn, Gln, Arg, Ser, Thr, Val or Tyr; the amino acidat position 560 is Cys, Ala, Phe, Gly, Ile, Met, Asn, Arg, Ser, Thr orVal; the amino acid at position 561 is Lys, Ala, Cys, Asp, Glu, Phe,Gly, His, lie, Leu, Met, Asn, Arg, Ser, Thr, Val or Tyr; the amino acidat position 562 is Asn, Cys, Asp, Glu, Gly, His, Leu, Met, Arg, Ser,Thr, Val or Tyr; the amino acid at position 563 is Val, Ala, Cys, Asp,Phe, His, Ile, Leu, Met, Asn, Gln, Thr or Trp; the amino acid atposition 564 is Ala, Cys, Gly, Met, Gln, Ser, Thr, Val, Trp or Tyr; theamino acid at position 571 is Ser, Thr or Cys; the amino acid atposition 575 is Val, Leu, Ile, Asp or Glu; the amino acid at position577 is Met, Leu, Val or Ile; the amino acid at position 579 is Glu, Asp,Asn or Gln; the amino acid at position 583 is Asp or Glu; the amino acidat position 589 is Met, Ile, Val or Leu; the amino acid at position 590is Met, Ile, Val or Leu; the amino acid at position 593 is Met, Leu, Valor Ile; the amino acid at position 595 is Arg, Lys, Asn or Gln; theamino acid at position 596 is Ser or Thr; the amino acid at position 597is Gln, Asn or His; the amino acid at position 607 is Ala, Gly, Ile, Leuor Val; the amino acid at position 608 is Asp, Glu, Gln or Asn; theamino acid at position 612 is Tyr, His or Phe; the amino acid atposition 617 is Thr, Ser, Leu, Val or Ile; the amino acid at position618 is Gln, Asn or His; the amino acid at position 625 is Arg, Lys, Thror Ser; the amino acid at position 626 is Met, Leu, Val or Ile; theamino acid at position 628 is Leu, Val or Ile; the amino acid atposition 633 is Ile, Leu, Val or Met; the amino acid at position 634 isLeu, Ile, Val or Met; the amino acid at position 642 is Arg, Lys or Met;the amino acid at position 646 is Leu, Ala, Cys, Gly, Ile, Met, Asn,Gln, Ser, Thr or Val; the amino acid at position 647 is Leu, Asp, Gly,Met, Asn, Gln or Thr; the amino acid at position 648 is Met, Ala, Cys,Asp, Glu, Phe, Gly, His, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Val,Trp or Tyr; the amino acid at position 649 is Pro, Ala, Cys, Asp, Glu,Phe, Gly, His, Lys, Met, Asn, Gln, Arg, Ser, Thr, Trp or Tyr; the aminoacid at position 650 is Thr, Ala, Cys, Asp, Phe, Gly, His, Ile, Lys,Leu, Met, Pro, Gln, Arg, Ser, Val or Tyr; the amino acid at position 651is Glu, Ala, Cys, Asp, Gly, His, Ile, Leu, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val or Tyr; the amino acid at position 652 is Leu, Cys, Phe, lie,Lys, Met, Pro, Arg, Ser, Thr or Val; the amino acid at position 653 isThr, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Arg, Ser, Val orTrp; the amino acid at position 654 is Thr, Ala, Cys, Phe, Ile, Lys,Leu, Met, Pro, Arg, Ser, Val, Trp or Tyr; the amino acid at position 655is Trp, Phe or Tyr; the amino acid at position 658 is Gly, Lys, Ala orArg; the amino acid at position 663 is Gly or Ala; the amino acid atposition 664 is Asp, Glu, Gln or Asn; the amino acid at position 668 isAla, Gly, Ser or Thr; the amino acid at position 669 is Gln, Asn or His;the amino acid at position 671 is Asn, Gln, Thr or Ser the amino acid atposition 675 is Ile, Val, Ile, Thr or Ser; the amino acid at position678 is Met, Ile, Ala, Leu, Ser or Thr; the amino acid at position 682 isPro, Asn or Gln; the amino acid at position 683 is Ser, Thr or Pro; theamino acid at position 685 is Asp, Glu, Asp or Asn; the amino acid atposition 694 is Asp, Glu, Ala or Gly; the amino acid at position 697 isAsn, Gln, Thr or Ser; the amino acid at position 704 is Glu, Asp, Ala orGly; the amino acid at position 714 is Ala or Gly; the amino acid atposition 721 is Ser, Thr or Phe; the amino acid at position 722 is Ser,Thr, Gln or Asn; the amino acid at position 724 is Ser or Thr; the aminoacid at position 734 is His, Asn or Gln; the amino acid at position 736is Val, Leu, lie or Ala; the amino acid at position 737 is Lys, Arg, Asnor Gln; the amino acid at position 739 is Ala, Gly, Thr or Ser; theamino acid at position 740 is Ser, Thr or Met; the amino acid atposition 741 is Gly, Ala, Gln or Asn; the amino acid at position 742 isIle, Leu, Val, Ala or Gly; the amino acid at position 743 is Gly ordeleted; the amino acid at position 745 is Gly, Ala, Glu or Asp; theamino acid at position 751 is Thr, Ser, Gly or Ala; the amino acid atposition 753 is Gln, Asn, Lys or Arg; the amino acid at position 754 isThr or Ser; the amino acid at position 756 is Thr, Ser, Leu, Val or Ile;the amino acid at position 757 is Val, Leu or Ile; the amino acid atposition 766 is Ile, Leu or Val; the amino acid at position 771 is Arg,Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Ser, Thr, Val, Trp orTyr; the amino acid at position 772 is Arg, Ala, Cys, Asp, Glu, Phe,Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Ser, Thr, Val, Trp or Tyr; theamino acid at position 773 is Asp, Ala, Glu, Phe, Gly, His, Ile, Lys,Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr; the amino acid atposition 774 is Gln, Ala, Asp, Gly, His, Ile, Lys, Leu, Met, Asn, Pro,Arg, Ser, Thr, Val, Trp or Tyr; the amino acid at position 775 is Val,Ala, Cys, Asp, Glu, Gly, His, Ile, Asn, Pro, Gln, Arg, Ser, Thr or Tyr;the amino acid at position 776 is Leu, Ala, Cys, Asp, Glu, Phe, Gly,His, Ile, Lys, Asn, Pro, Gln, Arg, Ser, Thr, Val or Tyr; the amino acidat position 777 is Pro, Ala, Cys, Asp, Glu, Phe, Gly, His, Lys, Leu,Met, Asn, Gln, Ser, Thr, Val, Trp or Tyr; the amino acid at position 778is Phe, Ala, His, Ile, Leu, Met, Asn, Gin, Ser, Val, Trp or Tyr; theamino acid at position 779 is Gln, Ala, Cys, Asp, Glu, Gly, His, Lys,Leu, Asn, Pro, Arg, Ser, Thr or Val; the amino acid at position 780 isAla, Cys, Asn, Pro, Gin or Ser; the amino acid at position 781 is Ala,Cys, Asp, Glu, Phe, Gly, His, Ile, Asn, Gln, Arg, Ser, Thr, Val, Trp orTyr; the amino acid at position 782 is Ala, Cys, Asp, Glu, Phe, Gly,His, Ile, Lys, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr; the aminoacid at position 783 is Pro, Ala, Cys, Asp, Glu, Gly, His, Asn, Gln,Arg, Ser, Thr or Val; the amino acid at position 784 is Leu, Ala, Glu,Phe, His, Ile, Lys, Met, Asn, Pro, Gln, Ser, Thr, Val or Trp; the aminoacid at position 785 is Asn, Ala, Cys, Glu, Phe, Gly, His, Ile, Lys,Leu, Met, Gln, Arg, Ser, Thr, Val, Trp or Tyr; the amino acid atposition 786 is Tyr, Phe, Ile, Leu or Trp; the amino acid at position787 is His, Asn or Gln; the amino acid at position 788 is Tyr or Met;the amino acid at position 789 is Ala, Lys or Arg; the amino acid atposition 790 is Tyr or Thr; the amino acid at position 791 is Arg, Lys,Gly or Ala; the amino acid at position 792 is Leu, Ile, Val, Thr or Ser;the amino acid at position 796 is Asp or Glu; the amino acid at position797 is Ser, Thr or Ala the amino acid at position 802 is Glu, Lys, Asp,Asn or Gln; the amino acid at position 806 is Gln, Asp, Glu, Asn or His;the amino acid at position 810 is Lys, Arg or Thr; the amino acid atposition 819 is Arg, Lys or His; the amino acid at position 829 is Lys,Ser, Ala or Pro; the amino acid at position 832 is Ala, Lys, Arg, Asp orGlu; the amino acid at position 833 is Gly, Ala, Asp or Glu; the aminoacid at position 842 is Leu, Ile, Val or Pro; the amino acid at position847 is Gln, Asn, Asp or Glu; the amino acid at position 848 is Ile, Leuor Val; the amino acid at position 849 is Val, Leu, Ile, Gly or Ala; theamino acid at position 855 is Thr, Ser or Met; the amino acid atposition 860 is Ile, Leu or Val; and the amino acid at position 864 isHis, Asn or Gln.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a fern species in the Division Pteridophyta.The phylogeny of ferns as used herein is based on the classification forextant ferns by A. R. Smith et al, TAXON, 55:705-731 (2006). Theconsensus phylogeny based on the classification by A. R. Smith is shownin FIG. 1. Other phylogenic classifications of extant ferns are known toone skilled in the art. Additional information on the phylogeny of fernscan be found at mobot.org/MOBOT/research/APweb/(which can be accessedusing the “www” prefix) and Schuettpelz E. and Pryer K. M., TAXON 56:1037-1050 (2007) based on three plastid genes. Additional fern and otherprimitive plant species can be found athomepages.caverock.net.nz/˜bj/fern/list.htm (which can be accessed usingthe http:// prefix).

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a fern species in the Class Psilotopsida. Insome embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a fern species in the Class Psilotopsida,Order Psilotales. In some embodiments the the nucleic acid moleculeencoding PtIP-83 polypeptide is derived from a fern species in the ClassPsilotopsida, Order Ophioglossales. In some embodiments the nucleic acidmolecule encoding the PtIP-83 polypeptide is derived from a fern speciesin the Class Psilotopsida, Order Ophioglossales, Family Psilotaceae. Insome embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a fern species in the Class Psilotopsida,Order Ophioglossales Family Ophioglossaceae. In some embodiments thenucleic acid molecule encoding the PtIP-83 polypeptide is derived from afern species in the Genus Ophioglossum L., Botrychium, Botrypus,Helminthostachys, Ophioderma, Cheiroglossa, Sceptridium or Mankyua.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a species in the ClassPolypodiopsida/Pteridopsida. In some embodiments the nucleic acidmolecule encoding the PtIP-83 polypeptide is derived from a fern speciesin the Order Osmundales (royal ferns); Family Osmundaceae. In someembodiments the nucleic acid molecule encoding the PtIP-83 polypeptideis derived from a fern species in the Order Hymenophyllales; FamilyHymenophyllaceae. In some embodiments the nucleic acid molecule encodingthe PtIP-83 polypeptide is derived from a fern species in the OrderGleicheniales; Family Gleicheniaceae, Family Dipteridaceae or FamilyMatoniaceae. In some embodiments the nucleic acid molecule encoding thePtIP-83 polypeptide is derived from a fern species in the OrderSchizaeales; Family Lygodiaceae, Family Anemiaceae or FamilySchizaeaceae. In some embodiments the nucleic acid encoding the PtIP-83polypeptide is derived from a fern species in the Order Schizaeales;Family Schizaeaceae, Genus Lygodium selected from but not limited toLygodium articulatum, Lygodium circinatum, Lygodium conforme, Lygodiumcubense, Lygodium digitatum, Lygodium flexuosum, Lygodium heterodoxum,Lygodium japonicum, Lygodium kerstenii, Lygodium lanceolatum, Lygodiumlongifolium, Lygodium merrilii, Lygodium micans, Lygodium microphyllum,Lygodium microstachyum, Lygodium oligostachyum, Lygodium palmatum,Lygodium polystachyum, Lygodium radiatum, Lygodium reticulatum, Lygodiumsalicifolium, Lygodium scandens, Lygodium smithianum, Lygodiumsubareolatum, Lygodium trifurcatum, Lygodium venustum, Lygodiumversteeghii, Lygodium volubile, and Lygodium yunnanense. In someembodiments the nucleic acid molecule encoding the PtIP-83 polypeptideis derived from a fern species in the Order Salviniales; FamilyMarsileaceae or Family Salviniaceae. In some embodiments the nucleicacid molecule encoding the PtIP-83 polypeptide is derived from a fernspecies in the Order Cyatheales; Family Thyrsopteridaceae, FamilyLoxsomataceae, Family Culcitaceae, Family Plagiogyriaceae, FamilyCibotiaceae, Family Cyatheaceae, Family Dicksoniaceae or FamilyMetaxyaceae.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a fern species in the Order Polypodiales;Family Lindsaeaceae, Family Saccolomataceae, Family Cystodiaceae, FamilyDennstaedtiaceae, Family Pteridaceae, Family Aspleniaceae, FamilyThelypteridaceae, Family Woodsiaceae, Family Onocleaceae, FamilyBlechnaceae, Family Dryopteridaceae, Family Lomariopsidaceae, FamilyTectariaceae, Family Oleandraceae, Family Davalliaceae or FamilyPolypodiaceae.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a fern species in the Order Polypodiales,Family Pteridaceae, Genus Adiantaceae selected from but not limited toAdiantum aethiopicum, Adiantum aleuticum, Adiantum bonatianum, Adiantumcajennense, Adiantum capillus-junonis, Adiantum capillus-veneris,Adiantum caudatum, Adiantum chienii, Adiantum chilense, Adiantumcuneatum, Adiantum cunninghamii, Adiantum davidii, Adiantum diaphanum,Adiantum edentulum, Adiantum edgeworthii, Adiantum excisum, Adiantumfengianum, Adiantum fimbriatum, Adiantum flabellulatum, Adiantumformosanum, Adiantum formosum, Adiantum fulvum, Adiantum gravesii,Adiantum hispidulum, Adiantum induratum, Adiantum jordanii, Adiantumjuxtapositum, Adiantum latifolium, Adiantum leveillei, Adiantumlianxianense, Adiantum malesianum, Adiantum mariesii, Adiantummonochlamys, Adiantum myriosorum, Adiantum obliquum, Adiantumogasawarense, Adiantum pedatum, Adiantum pentadactylon, Adiantumperuvianum, Adiantum philippense, Adiantum princeps, Adiantum pubescens,Adiantum raddianum, Adiantum reniforme, Adiantum roborowskii, Adiantumserratodentatum, Adiantum sinicum, Adiantum soboliferum, Adiantumsubcordatum, Adiantum tenerum, Adiantum terminatum, Adiantumtetraphyllum, Adiantum trapeziforme, Adiantum venustum, Adiantumviridescens, and Adiantum viridimontanum.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a fern species in the Order Polypodiales,Family Aspleniaceae, Genus Asplenium.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a fern species in the Order Polypodiales,Family Aspleniaceae, Genus Asplenium selected from but not limited toAsplenium adiantum, Asplenium adulterinum, Asplenium aequibasis,Asplenium aethiopicum, Asplenium africanum, Asplenium×alternifolium,Asplenium angustum, Asplenium antiquum, Asplenium ascensionis, Aspleniumattenuatum, Asplenium aureum, Asplenium auritum, Aspleniumaustralasicum, Asplenium azoricum, Asplenium bifrons, Aspleniumbillottii, Asplenium bipinnatifidum, Asplenium brachycarpum, Aspleniumbradleyi, Asplenium bulbiferum, Asplenium caudatum, Asplenium ceterach,Asplenium compressum, Asplenium congestum, Asplenium corderoanum,Asplenium crinicaule, Asplenium cristatum, Asplenium cuneifolium,Asplenium cymbifolium, Asplenium daghestanicum, Asplenium dalhousiae,Asplenium dareoides, Asplenium daucifolium, Asplenium difforme,Asplenium fissum, Asplenium dimorphum, Asplenium divaricatum, Aspleniumdregeanum, Asplenium×ebenoides, Asplenium ecuadorense, Asplenium feeiKunze, Asplenium fissum, Asplenium flabellifolium, Asplenium flaccidum,Asplenium fontanum, Asplenium forisiense, Asplenium formosum, Aspleniumgemmiferum, Asplenium×germanicum, Asplenium gueinzii, Asplenium goudeyi,Asplenium hemionitis, Asplenium hermannii-christii, Aspleniumhookerianum, Asplenium hybridum, Asplenium incisum, Asplenium×jacksonii,Asplenium×kenzoi, Asplenium laciniatum, Asplenium lamprophyllum,Asplenium laserpitiifolium, Asplenium lepidum, Asplenium listeri,Asplenium longissimum, Asplenium lucidum, Asplenium lunulatum, Aspleniumlyallii, Asplenium macedonicum, Asplenium majoricum, Asplenium marinum,Asplenium×microdon, Asplenium milnei, Asplenium montanum, Aspleniummusifolium, Asplenium nidus, Asplenium normale, Asplenium obliquum,Asplenium oblongifolium, Asplenium obovatum, Asplenium obtusatum,Asplenium oligolepidum, Asplenium oligophlebium, Asplenium onopteris,Asplenium pacificum, Asplenium paleaceum, Asplenium palmeri, Aspleniumpetrarchae, Asplenium pinnatifidum, Asplenium planicaule, Aspleniumplatybasis, Asplenium platyneuron, Asplenium polyodon, Aspleniumpraemorsum, Asplenium prolongatum, Asplenium pteridoides, Aspleniumresiliens, Asplenium rhizophyllum, Asplenium richardii, Aspleniumruprechtii, Asplenium ruta-muraria, Asplenium rustifolium, Aspleniumsagittatum, Asplenium sandersonii, Asplenium×sarniense, Aspleniumschizotrichum, Asplenium schweinfurthii, Asplenium scleroprium,Asplenium scolopendrium (syn. Phyllitis scolopendrium), Aspleniumseelosii, Asplenium septentrionale, Aspleniumseptentrionale×trichomanes, Asplenium serra, Asplenium serratum,Asplenium sessilifolium, Asplenium shuttleworthianum, Aspleniumsimplicifrons, Asplenium splendens, Asplenium surrogatum, Aspleniumtenerum, Asplenium terrestre, Asplenium theciferum, Aspleniumthunbergii, Asplenium trichomanes, Asplenium tutwilerae, Aspleniumvespertinum, Asplenium vieillardii, Asplenium virens, Asplenium viride,Asplenium vittiforme, and Asplenium viviparum.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a fern species in the Order Polypodiales,Family Blechnaceae, Genus Blecnum.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a fern species in the Order Polypodiales,Family Dryopteridaceae Genus Acrophorus, Genus Acrorumohra, GenusAnapausia, Genus Arachniodes, Genus Bolbitis, Genus Ctenitis, GenusCyclodium, Genus Cyrtogonellum, Genus Cyrtomidictyum, Genus Cyrtomium,Genus Diacalpe, Genus Didymochlaena, Genus Dryopsis, Genus Dryopteris,Genus Elaphoglossum, Genus Hypodematium, Genus Lastreopsis, GenusLeptorumohra, Genus Leucostegia, Genus Lithostegia, Genus Lomagramma,Genus Maxonia, Genus Megalastrum, Genus Olfersia, Genus Peranema, GenusPhanerophlebia, Genus Phanerophlebiopsis, Genus Polybotrya, GenusPolystichopsis, Genus Polystichum, Genus Rumohra, Genus Sorolepidium,Genus Stigmatopteris or Genus Teratophyllum.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a fern species in the Order Polypodiales,Family Dryopteridaceae, Genus Polystichum. In some embodiments thenucleic acid molecule encoding the PtIP-83 polypeptide is derived from afern species in the Order Polypodiales, Family Dryopteridaceae, GenusPolystichum selected from but not limited to Polystichum acanthophyllum,Polystichum acrostichoides, Polystichum aculeatum, Polystichumacutidens, Polystichum acutipinnulum, Polystichum alcicorne, Polystichumaleuticum, Polystichum andersonii, Polystichum atkinsonii, Polystichumaustraliense, Polystichum bakerianum, Polystichum biaristatum,Polystichum bomiense, Polystichum bonseyi, Polystichum brachypterum,Polystichum braunii, Polystichum brachypterum, Polystichum calderonense,Polystichum californicum, Polystichum capillipes, Polystichum castaneum,Polystichum chilense, Polystichum christii Ching, Polystichum chuniiChing, Polystichum craspedosorum, Polystichum cyclolobum, Polystichumcystostegia, Polystichum deltodon, Polystichum dielsii, Polystichumdiscretum, Polystichum drepanum, Polystichum dudleyi, Polystichumduthiei, Polystichum echinatum, Polystichum erosum, Polystichumexcellens, Polystichum eximium, Polystichum falcatipinnum, Polystichumfalcinellum, Polystichum fallax, Polystichum formosanum, Polystichumgongboense, Polystichum grandifrons, Polystichum gymnocarpium,Polystichum haleakalense, Polystichum hancockii, Polystichumhecatopteron, Polystichum herbaceum, Polystichum imbricans, Polystichumincongruum, Polystichum kruckebergii, Polystichum kwakiutlii,Polystichum lachenense, Polystichum lanceolatum, Polystichum lemmonii,Polystichum lentum, Polystichum lonchitis, Polystichum longidens,Polystichum longipaleatum, Polystichum longipes, Polystichum luctuosum,Polystichum macleae, Polystichum macrochlaenum, Polystichum makinoi,Polystichum martini, Polystichum mayebarae, Polystichum mediocre,Polystichum medogense, Polystichum microchlamys, Polystichum mohrioides,Polystichum mollissimum, Polystichum monticola, Polystichum moorei,Polystichum morii, Polystichum moupinense, Polystichum muricatum,Polystichum nakenense, Polystichum neolobatum, Polystichum nepalense,Polystichum ningshenense, Polystichum obliquum, Polystichum omeiense,Polystichum ordinatum, Polystichum orientalitibeticum, Polystichumparamoupinense, Polystichum parvipinnulum, Polystichum piceopaleaceum,Polystichum polyblepharum, Polystichum prescottianum, Polystichumprionolepis, Polystichum proliferum, Polystichum pseudocastaneum,Polystichum pseudomakinoi, Polystichum punctiferum, Polystichum pungens,Polystichum qamdoense, Polystichum retrosopaleaceum, Polystichumrhombiforme, Polystichum rhomboidea, Polystichum richardii, Polystichumrigens, Polystichum rotundilobum, Polystichum scopulinum, Polystichumsemifertile, Polystichum setiferum, Polystichum setigerum, Polystichumshensiense, Polystichum silvaticum, Polystichum simplicipinnum,Polystichum sinense, Polystichum squarrosum, Polystichum stenophyllum,Polystichum stimulans, Polystichum submite, Polystichum tacticopterum,Polystichum thomsoni, Polystichum tibeticum, Polystichum transvaalense,Polystichum tripteron, Polystichum tsus-simense, Polystichum vestitum,Polystichum wattii, Polystichum whiteleggei, Polystichum xiphophyllum,Polystichum yadongense, and Polystichum yunnanense.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a fern species in the Order Polypodiales,Family Dryopteridaceae, Genus Rumohra. In some embodiments the nucleicacid molecule encoding the PtIP-83 polypeptide is derived from a fernspecies in the Order Polypodiales, Family Dryopteridaceae, Genus Rumohraselected from but not limited to Rumohra adiantiformis, Rumohraaristata, Rumohra bartonae, Rumohra berteroana, Rumohra capuronii,Rumohra glandulosa, Rumohra humbertii, Rumohra linearisquamosa, Rumohralokohensis, Rumohra madagascarica, and Rumohra quadrangularis.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a fern species in the Order Polypodiales,Family Polypodiaceae Genus Campyloneurum, Genus Drynaria, GenusLepisorus, Genus Microgramma, Genus Microsorum, Genus Neurodium, GenusNiphidium, Genus Pecluma M.G., Genus Phlebodium, Genus Phymatosorus,Genus Platycerium, Genus Pleopeltis, Genus Polypodium.

In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Order Polypodiales, Family Polypodiaceae, GenusMicrosorum.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a fern species in the Order Polypodiales,Family Polypodiaceae, Genus Microsorum. selected from but not limited toMicrosorum alatum, Microsorum angustifolium, Microsorum aurantiacum,Microsorum australiense, Microsorum baithoense, Microsorum basicordatum,Microsorum biseriatum, Microsorum brassii, Microsorum buergerianum,Microsorum chapaense, Microsorum cinctum, Microsorum commutatum,Microsorum congregatifolium, Microsorum cuneatum, Microsorum cuspidatum,Microsorum dengii, Microsorum egregium, Microsorum emeiensis, Microsorumensatum, Microsorum ensiforme, Microsorum excelsum, Microsorum fortunei,Microsorum griseorhizoma, Microsorum grossum, Microsorum hemionitideum,Microsorum henryi, Microsorum heterocarpum, Microsorum heterolobum,Microsorum howense, Microsorum insigne, Microsorum intermedium,Microsorum kongtingense, Microsorum krayanense, Microsorum lanceolatum,Microsorum lancifolium, Microsorum lastii, Microsorum latilobatum,Microsorum leandrianum, Microsorum lineare, Microsorum linguiforme,Microsorum longissimum, Microsorum longshengense, Microsorum maculosum,Microsorum maximum, Microsorum membranaceum, Microsorum membranifolium,Microsorum microsorioides, Microsorum minor, Microsorum monstrosum,Microsorum muliense, Microsorum mutense, Microsorum nanchuanense,Microsorum ningpoense, Microsorum normale, Microsorum novae-zealandiae,Microsorum ovalifolium, Microsorum ovatum, Microsorum palmatopedatum,Microsorum pappei, Microsorum papuanum, Microsorum parksii, Microsorumpentaphyllum, Microsorum piliferum, Microsorum pitcairnense, Microsorumpowellii, Microsorum pteropodum, Microsorum pteropus, Microsorumpunctatum, Microsorum pustulatum, Microsorum rampans, Microsorumrevolutum, Microsorum rubidum, Microsorum samarense, Microsorumsapaense, Microsorum sarawakense, Microsorum scandens, Microsorumscolopendria, Microsorum sibomense, Microsorum sinense, Microsorumsopuense, Microsorum spectrum, Microsorum steerei, Microsorumsubhemionitideum, Microsorum submarginale, Microsorum subnudum,Microsorum superficiale, Microsorum takhtajanii, Microsorum tenuipes,Microsorum tibeticum, Microsorum triglossum, Microsorum truncatum,Microsorum tsaii, Microsorum varians, Microsorum venosum, Microsorumvieillardii, Microsorum×inaequibasis, Microsorum yiliangensis, andMicrosorum zippelii.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a fern species in the Order Polypodiales,Family Polypodiaceae, Genus Polypodium L. In some embodiments thenucleic acid molecule encoding the PtIP-83 polypeptide is derived from afern species in the Order Polypodiales, Family Polypodiaceae, GenusPolypodium L. selected from but not limited to Polypodium absidatum,Polypodium acutifolium, Polypodium adiantiforme, Polypodium aequale,Polypodium affine, Polypodium albidopaleatum, Polypodium alcicorne,Polypodium alfarii, Polypodium alfredii, Polypodium alfredii var.curtii, Polypodium allosuroides, Polypodium alsophilicola, Polypodiumamamianum, Polypodium amoenum, Polypodium amorphum, Polypodiumanetioides, Polypodium anfractuosum, Polypodium anguinum, Polypodiumangustifolium f. remotifolia, Polypodium angustifolium var. amphostenon,Polypodium angustifolium var. heterolepis, Polypodium angustifolium var.monstrosa, Polypodium angustipaleatum, Polypodium angustissimum,Polypodium anisomeron var. pectinatum, Polypodium antioquianum,Polypodium aoristisorum, Polypodium apagolepis, Polypodium apicidens,Polypodium apiculatum, Polypodium apoense, Polypodium appalachianum,Polypodium appressum, Polypodium arenarium, Polypodium argentinum,Polypodium argutum, Polypodium armatum, Polypodium aromaticum,Polypodium aspersum, Polypodium assurgens, Polypodium atrum, Polypodiumauriculatum, Polypodium balaonense, Polypodium balliviani, Polypodiumbamleri, Polypodium bangii, Polypodium bartlettii, Polypodium basale,Polypodium bernoullii, Polypodium biauritum, Polypodium bifrons,Polypodium blepharodes, Polypodium bolivari, Polypodium bolivianum,Polypodium bolobense, Polypodium bombycinum, Polypodium bombycinum var.insularum, Polypodium bradeorum, Polypodium bryophilum, Polypodiumbryopodum, Polypodium buchtienii, Polypodium buesii, Polypodiumbulbotrichum, Polypodium caceresii, Polypodium californicum f.brauscombii, Polypodium californicum f. parsonsiae, Polypodiumcalifornicum, Polypodium calophlebium, Polypodium calvum, Polypodiumcamptophyllarium var. abbreviatum, Polypodium capitellatum, Polypodiumcarpinterae, Polypodium chachapoyense, Polypodium chartaceum, Polypodiumchimantense, Polypodium chiricanum, Polypodium choquetangense,Polypodium christensenii, Polypodium christii, Polypodium chrysotrichum,Polypodium ciliolepis, Polypodium cinerascens, Polypodium collinsii,Polypodium colysoides, Polypodium confluens, Polypodium conforme,Polypodium confusum, Polypodium congregatifolium, Polypodium connellii,Polypodium consimile var. bourgaeanum, Polypodium consimile var. minor,Polypodium conterminans, Polypodium contiguum, Polypodium cookii,Polypodium coriaceum, Polypodium coronans, Polypodium costaricense,Polypodium costatum, Polypodium crassifolium f. angustissimum,Polypodium crassifolium var. longipes, Polypodium crassulum, Polypodiumcraterisorum, Polypodium cryptum, Polypodium crystalloneuron, Polypodiumcucullatum var. planum, Polypodium cuencanum, Polypodium cumingianum,Polypodium cupreolepis, Polypodium curranii, Polypodium curvans,Polypodium cyathicola, Polypodium cyathisorum, Polypodium cyclocolpon,Polypodium daguense, Polypodium damunense, Polypodium dareiformioides,Polypodium dasypleura, Polypodium decipiens, Polypodium decorum,Polypodium delicatulum, Polypodium deltoideum, Polypodium demeraranum,Polypodium denticulatum, Polypodium diaphanum, Polypodium dilatatum,Polypodium dispersum, Polypodium dissectum, Polypodium dissimulans,Polypodium dolichosorum, Polypodium dolorense, Polypodiumdonnell-smithii, Polypodium drymoglossoides, Polypodium ebeninum,Polypodium eggersii, Polypodium elmeri, Polypodium elongatum, Polypodiumenterosoroides, Polypodium erubescens, Polypodium erythrolepis,Polypodium erythrotrichum, Polypodium eurybasis, Polypodium eurybasisvar. villosum, Polypodium exornans, Polypodium falcoideum, Polypodiumfallacissimum, Polypodium farinosum, Polypodium faucium, Polypodiumfeei, Polypodium ferrugineum, Polypodium feuillei, Polypodium firmulum,Polypodium firmum, Polypodium flaccidum, Polypodium flagellare,Polypodium flexuosum, Polypodium flexuosum var. ekmanii, Polypodiumforbesii, Polypodium formosanum, Polypodium fraxinifolium subsp.articulatum, Polypodium fraxinifolium subsp. luridum, Polypodiumfructuosum, Polypodium fucoides, Polypodium fulvescens, Polypodiumgaleottii, Polypodium glaucum, Polypodium glycyrrhiza, Polypodiumgracillimum, Polypodium gramineum, Polypodium grandifolium, Polypodiumgratum, Polypodium graveolens, Polypodium griseo-nigrum, Polypodiumgriseum, Polypodium guttatum, Polypodium haalilioanum, Polypodiumhammatisorum, Polypodium hancockii, Polypodium haplophlebicum,Polypodium harrisii, Polypodium hastatum var. simplex, Polypodiumhawaiiense, Polypodium heanophyllum, Polypodium helleri, Polypodiumhemionitidium, Polypodium henryi, Polypodium herzogii, Polypodiumhesperium, Polypodium hessii, Polypodium hombersleyi, Polypodiumhostmannii, Polypodium humile, Polypodium hyalinum, Polypodium iboense,Polypodium induens var. subdentatum, Polypodium insidiosum, Polypodiuminsigne, Polypodium intermedium subsp. masafueranum var. obtuseserratum,Polypodium intramarginale, Polypodium involutum, Polypodium itatiayense,Polypodium javanicum, Polypodium juglandifolium, Polypodium kaniense,Polypodium knowltoniorum, Polypodium kyimbilense, Polypodiuml'herminieri var. costaricense, Polypodium lachniferum f. incurvata,Polypodium lachniferum var. glabrescens, Polypodium lachnopus,Polypodium lanceolatum var. complanatum, Polypodium lanceolatum var.trichophorum, Polypodium latevagans, Polypodium laxifrons, Polypodiumlaxifrons var. lividum, Polypodium lehmannianum, Polypodium leiorhizum,Polypodium leptopodon, Polypodium leuconeuron var. angustifolia,Polypodium leuconeuron var. latifolium, Polypodium leucosticta,Polypodium limulum, Polypodium lindigii, Polypodium lineatum, Polypodiumlomarioides, Polypodium longifrons, Polypodium loretense, Polypodiumloriceum var. umbraticum, Polypodium loriforme, Polypodium loxogramme f.gigas, Polypodium ludens, Polypodium luzonicum, Polypodium lycopodioidesf. obtusum, Polypodium lycopodioides L., Polypodium macrolepis,Polypodium macrophyllum, Polypodium macrosorum, Polypodiummacrosphaerum, Polypodium maculosum, Polypodium madrense, Polypodiummanmeiense, Polypodium margaritiferum, Polypodium maritimum, Polypodiummartensii, Polypodium mayoris, Polypodium megalolepis, Polypodiummelanotrichum, Polypodium menisciifolium var. pubescens, Polypodiummeniscioides, Polypodium merrillii, Polypodium mettenii, Polypodiummexiae, Polypodium microsorum, Polypodium militare, Polypodium minimum,Polypodium minusculum, Polypodium mixtum, Polypodium mollendense,Polypodium mollissimum, Polypodium moniliforme var. minus, Polypodiummonoides, Polypodium monticola, Polypodium montigenum, Polypodiummoritzianum, Polypodium moultonii, Polypodium multicaudatum, Polypodiummultilineatum, Polypodium multisorum, Polypodium munchii, Polypodiummuscoides, Polypodium myriolepis, Polypodium myriophyllum, Polypodiummyriotrichum, Polypodium nematorhizon, Polypodium nemorale, Polypodiumnesioticum, Polypodium nigrescentium, Polypodium nigripes, Polypodiumnigrocinctum, Polypodium nimbatum, Polypodium nitidissimum, Polypodiumnitidissimum var. latior, Polypodium nubrigenum, Polypodium oligolepis,Polypodium oligosorum, Polypodium oligosorum, Polypodium olivaceum,Polypodium olivaceum var. elatum, Polypodium oodes, Polypodiumoosphaerum, Polypodium oreophilum, Polypodium ornatissimum, Polypodiumornatum, Polypodium ovatum, Polypodium oxylobum, Polypodium oxypholis,Polypodium pakkaense, Polypodium pallidum, Polypodium palmatopedatum,Polypodium palmeri, Polypodium panamense, Polypodium parvum, Polypodiumpatagonicum, Polypodium paucisorum, Polypodium pavonianum, Polypodiumpectinatum var. caliense, Polypodium pectinatum var. hispidum,Polypodium pellucidum, Polypodium pendulum var. boliviense, Polypodiumpercrassum, Polypodium perpusillum, Polypodium peruvianum var.subgibbosum, Polypodium phyllitidis var. elongatum, Polypodiumpichinchense, Polypodium pilosissimum, Polypodium pilosissimum var.glabriusculum, Polypodium pilossimum var. tunguraquensis, Polypodiumpityrolepis, Polypodium platyphyllum, Polypodium playfairii, Polypodiumplebeium var. cooperi, Polypodium plectolepidioides, Polypodiumpleolepis, Polypodium plesiosorum vari, Polypodium podobasis, Polypodiumpodocarpum, Polypodium poloense, Polypodium polydatylon, Polypodiumpolypodioides var. aciculare, Polypodium polypodioides var.michauxianum, Polypodium praetermissum, Polypodium preslianum var.immersum, Polypodium procerum, Polypodium procerum, Polypodiumproductum, Polypodium productum, Polypodium prolongilobum, Polypodiumpropinguum, Polypodium proteus, Polypodium pruinatum, Polypodiumpseudocapillare, Polypodium pseudofraternum, Polypodium pseudonutans,Polypodium pseudoserratum, Polypodium pulcherrimum, Polypodiumpulogense, Polypodium pungens, Polypodium purpusii, Polypodium radicale,Polypodium randallii, Polypodium ratiborii, Polypodium reclinatum,Polypodium recreense, Polypodium repens var. abruptum, Polypodiumrevolvens, Polypodium rhachipterygium, Polypodium rhomboideum,Polypodium rigens, Polypodium robustum, Polypodium roraimense,Polypodium roraimense, Polypodium rosei, Polypodium rosenstockii,Polypodium rubidum, Polypodium rudimentum, Polypodium rusbyi, Polypodiumsablanianum, Polypodium sarmentosum, Polypodium saxicola, Polypodiumschenckii, Polypodium schlechteri, Polypodium scolopendria, Polypodiumscolopendria, Polypodium scolopendrium, Polypodium scouleri, Polypodiumscutulatum, Polypodium segregatum, Polypodium semihirsutum, Polypodiumsemihirsutum var. fuscosetosum, Polypodium senile var. minor, Polypodiumsericeolanatum, Polypodium serraeforme, Polypodium serricula, Polypodiumsesquipedala, Polypodium sessilifolium, Polypodium setosum var. calvum,Polypodium setulosum, Polypodium shaferi, Polypodium sibomense,Polypodium siccum, Polypodium simacense, Polypodium simulans, Polypodiumsingeri, Polypodium sinicum, Polypodium sintenisii, Polypodium skutchii,Polypodium sloanei, Polypodium sodiroi, Polypodium sordidulum,Polypodium sordidum, Polypodium sphaeropteroides, Polypodium sphenodes,Polypodium sprucei, Polypodium sprucei var. furcativenosa, Polypodiumsteirolepis, Polypodium stenobasis, Polypodium stenolepis, Polypodiumstenopterum, Polypodium subcapillare, Polypodium subflabelliforme,Polypodium subhemionitidium, Polypodium subinaequale, Polypodiumsubintegrum, Polypodium subspathulatum, Polypodium subtile, Polypodiumsubvestitum, Polypodium subviride, Polypodium superficiale var.attenuatum, Polypodium superficiale var. chinensis, Polypodiumsursumcurrens, Polypodium tablazianum, Polypodium taenifolium,Polypodium tamandarei, Polypodium tatei, Polypodium tenuiculum var.acrosora, Polypodium tenuiculum var. brasiliense, Polypodium tenuilore,Polypodium tenuinerve, Polypodium tepuiense, Polypodium teresae,Polypodium tetragonum var. incompletum, Polypodium thysanolepis var.bipinnatifidum, Polypodium thyssanolepis, var. thyssanolepis, Polypodiumthyssanolepsi, Polypodium tobagense, Polypodium trichophyllum,Polypodium tridactylum, Polypodium tridentatum, Polypodium trifurcatumvar. brevipes, Polypodium triglossum, Polypodium truncatulum, Polypodiumtruncicola var. major, Polypodium truncicola var. minor, Polypodiumtuberosum, Polypodium tunguraguae, Polypodium turquinum, Polypodiumturrialbae, Polypodium ursipes, Polypodium vagans, Polypodiumvaldealatum, Polypodium versteegii, Polypodium villagranii, Polypodiumvirginianum f. cambroideum, Polypodium virginianum f. peraferens,Polypodium vittarioides, Polypodium vulgare, Polypodium vulgare L.,Polypodium vulgare subsp. oreophilum, Polypodium vulgare var.acuminatum, Polypodium vulpinum, Polypodium williamsii, Polypodiumwobbense, Polypodium×fallacissimum-guttatum, Polypodium xantholepis,Polypodium xiphopteris, Polypodium yarumalense, Polypodium yungense, andPolypodium zosteriforme.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a fern species in the Order Polypodiales,Family Polypodiaceae, Genus Platycerium.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a species in the Division Lycophyta. Thephylogeny of extant Lycopods as used herein is based on theclassification by N. Wikstrom, American Fern Journal, 91:150-156 (2001).Other phylogenic classifications of extant Lycopods are known to oneskilled in the art. Additional information on the phylogeny of ferns canbe found at mobot.org/MOBOT/research/APweb/ (which can be accessed usingthe “www” prefix) and Schuettpelz E. and Pryer K. M., TAXON 56:1037-1050 (2007) based on three plastid genes. Additional Lycopodspecies can be found at homepages.caverock.net.nz/˜bj/fern/list.htm(which can be accessed using the http:// prefix).

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a species in the Class Isoetopsida or ClassLycopodiopsida.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a species in the Class Isoetopsida, OrderSelaginales. In some embodiments the nucleic acid molecule encoding thePtIP-83 polypeptide is derived from a fern species in the ClassIsoetopsida, Order Selaginales, Family Selaginellaceae. In someembodiments the nucleic acid molecule encoding the PtIP-83 polypeptideis derived from a species in the Genus Selaginella.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a species in the Class Lycopodiopsida, OrderLycopodiales. In some embodiments the nucleic acid molecule encoding thePtIP-83 polypeptide is derived from a fern species in the ClassLycopodiopsida, Order Lycopodiales Family Lycopodiaceae or FamilyHuperziaceae. In some embodiments the nucleic acid molecule encoding thePtIP-83 polypeptide is derived from a species in the GenusAustrolycopodium, Dendrolycopodium, Diphasiastrum, Diphasium, Huperzia,Lateristachys, Lycopodiastrum, Lycopodiella, Lycopodium, Palhinhaea,Pseudodiphasium, Pseudolycopodiella, Pseudolycopodium or Spinulum. Insome embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a species in the Genus Lycopodium.

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprises an amino acid sequence MOTIF selected from: anamino acid sequence MOTIF 1 as represented by an amino acid sequence ofthe formulaMP[DE]MPSEADWSIFVNE[IV]EAVAEGMPTEVSEVP[AV]WKAKCKN[MV]AALGREM[SC]I (SEQID NO: 646); an amino acid sequence MOTIF 2 as represented by an aminoacid sequence of the formulaPQLQYRMYG[NS]LI[KN]QMAQVAQNYDQ[ED]FKQ[FL]KLFI[IA]QNQI[LF]GSYLLQQN[KR]A F(SEQ ID NO: 647); an amino acid sequence MOTIF 3 as represented by anamino acid sequence of the formulaNTFMQMTPFTRWRLRLSASASENA[EG]LAFPTATA[PL]DSTT[EQ][IV]VITFHVTAIR (SEQ IDNO: 648); an amino acid sequence MOTIF 4 as represented by an amino acidsequence of the formula [DN]FTSRHVVK[GD]IPVSLLLDGEDWEFEIPVQ[AG]GMSSFP(SEQ ID NO: 649); an amino acid sequence MOTIF 5 as represented by anamino acid sequence of the formulaIIHQP[SA]T[RQ][ST]G[IT]VYILLQGSTIFHDRRR[DE]EVMTFQAA[DA]PLN[FY][QH]YAYRLDTG (SEQ ID NO: 650); an amino acid sequence MOTIF 6 as represented by anamino acid sequence of the formulaS[HQ]ADRLAAIQP[AV]DLTN[HY]LEMAT[HQ]MDMRTT[RS][MI]L[IL]GLLN[MI]LRIQNAALMYEY (SEQ ID NO: 651); an amino acid sequence MOTIF 7 as represented by anamino acid sequence of the formula[VL]DRVEFSEVMVIHRMYVRL[SA]DL[ND]VGEL[PE]GA[EG][RK]VKR[VL]YV[FL]ADVVE(SEQ ID NO: 652); an amino acid sequence MOTIF 8 as represented by anamino acid sequence of the formulaA[DE]RELQMESFHSAVISQRRQEL[ND]TA[IF]AKM[DE]R[LM]SLQMEEE[NS]RAMEQAQKE M(SEQ ID NO: 653); an amino acid sequence MOTIF 9 as represented by anamino acid sequence of the formulaFVTAGATAPGA[AV]ASAGQAVSIAGQAAQ[AG]LRRVVEILE[GQ]LEAVMEVVAA[VI]K (SEQ IDNO: 654); an amino acid sequence MOTIF 10 as represented by an aminoacid sequence of the formulaDGMNWG[IT]YI[YH]GE[KE]V[EQ]RSPLLPSNAILAVWADRC[TI]ITSARHNH[VF]NAPGR[IV]I(SEQ ID NO: 655); an amino acid sequence MOTIF 11 as represented by anamino acid sequence of the formula[KV][VK][CA]RPPSPDM[MV]SAVAEHALWLNDVLLQVVQ[KN]ESQ[LM]QGT[AE]PYNECLAL LGR(SEQ ID NO: 656); an amino acid sequence MOTIF 12 as represented by anamino acid sequence of the formulaPTELT[VA]WPLGMDTV[AG]NLLIAQENAAL[VL]GLIQLGPSS (SEQ ID NO: 657); an aminoacid sequence MOTIF 13 as represented by an amino acid sequence of theformula RDQ[MT][HQ]MPGSVTVI[IV]LCRLLQFP[IT]DGSQA[TA]T (SEQ ID NO: 658);an amino acid sequence MOTIF 14 as represented by an amino acid sequenceof the formula TSIPVEVVTDP[SN]ILLGMQTTV[LH]IAEL (SEQ ID NO: 659); anamino acid sequence MOTIF as represented by an amino acid sequence ofthe formula EGLR[EQ]FQNRQVARA[VL]FAVLKAVA[MQ]I[AG] (SEQ ID NO: 660); anamino acid sequence MOTIF 16 as represented by an amino acid sequence ofthe formula W[TS]RVRIRHLEM[QH]F[AV]QEASG (SEQ ID NO: 661); an amino acidsequence MOTIF 17 as represented by an amino acid sequence of theformula QISELQY[ED]IWVQG[LM][ML]RDIA (SEQ ID NO: 662); an amino acidsequence MOTIF 18 as represented by an amino acid sequence of theformula TFTLGSGVTGITSMHGEPSLDPWNGVSLDSASPTAF (SEQ ID NO: 663); an aminoacid sequence MOTIF 19 as represented by an amino acid sequence of theformula MDYSTLYRDLNQIS (SEQ ID NO: 664); an amino acid sequence MOTIF 20as represented by an amino acid sequence of the formulaLRLPFM[QK]LHARVIEQN[VR]K[SE](SEQ ID NO: 665); an amino acid sequenceMOTIF 21 as represented by an amino acid sequence of the formulaVDSLEQVG[QH][IL]V[GD]AP (SEQ ID NO: 666); an amino acid sequence MOTIF22 as represented by an amino acid sequence of the formula[IV][EQ][CA]VMK[IM]GRF[VG][SL]VV (SEQ ID NO: 667); an amino acidsequence MOTIF 23 as represented by an amino acid sequence of theformula TLTNEPSE[EQ]F (SEQ ID NO: 668); and an amino acid sequence MOTIF24 as represented by an amino acid sequence of the formula LPRQSRNISF(SEQ ID NO: 669).

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprises an amino acid sequence MOTIF selected from: anamino acid sequence MOTIF 1 having at least 90% sequence identity to theamino acid sequence as represented by the formulaMP[DE]MPSEADWSIFVNE[IV]EAVAEGMPTEVSEVP[AV]WKAKCKN[MV]AALGREM[SC]I (SEQID NO: 646); an amino acid sequence MOTIF 2 having at least 90% sequenceidentity to the amino acid sequence as represented by the formulaPQLQYRMYG[NS]LI[KN]QMAQVAQNYDQ[ED]FKQ[FL]KLFI[IA]QNQI[LF]GSYLLQQN[KR]A F(SEQ ID NO: 647); an amino acid sequence MOTIF 3 having at least 90%sequence identity to the amino acid sequence as represented by theformula NTFMQMTPFTRWRLRLSASASENA[EG]LAFPTATA[PL]DSTT[EQ][IV]VITFHVTAIR(SEQ ID NO: 648); an amino acid sequence MOTIF 4 having at least 90%sequence identity to the amino acid sequence as represented by theformula [DN]FTSRHVVK[GD]IPVSLLLDGEDWEFEIPVQ[AG]GMSSFP (SEQ ID NO: 649);an amino acid sequence MOTIF 5 having at least 90% sequence identity tothe amino acid sequence as represented by the formulaIIHQP[SA]T[RQ][ST]G[IT]VYILLQGSTIFHDRRR[DE]EVMTFQAA[DA]PLN[FY][QH]YAYRLDTG (SEQ ID NO: 650); an amino acid sequence MOTIF 6 having at least 90%sequence identity to the amino acid sequence as represented by theformulaS[HQ]ADRLAAIQP[AV]DLTN[HY]LEMAT[HQ]MDMRTT[RS][MI]L[IL]GLLN[MI]LRIQNAALMYEY (SEQ ID NO: 651); an amino acid sequence MOTIF 7 having at least 90%sequence identity to the amino acid sequence as represented by theformula[VL]DRVEFSEVMVIHRMYVRL[SA]DL[ND]VGEL[PE]GA[EG][RK]VKR[VL]YV[FL]ADVVE(SEQ ID NO: 652); an amino acid sequence MOTIF 8 having at least 90%sequence identity to the amino acid sequence as represented by theformulaA[DE]RELQMESFHSAVISQRRQEL[ND]TA[IF]AKM[DE]R[LM]SLQMEEE[NS]RAMEQAQKE M(SEQ ID NO: 653); an amino acid sequence MOTIF 9 having at least 90%sequence identity to the amino acid sequence as represented by theformula FVTAGATAPGA[AV]ASAGQAVSIAGQAAQ[AG]LRRVVEILE[GQ]LEAVMEVVAA[VI]K(SEQ ID NO: 654); an amino acid sequence MOTIF 10 having at least 90%sequence identity to the amino acid sequence as represented by theformulaDGMNWG[IT]YI[YH]GE[KE]V[EQ]RSPLLPSNAILAVWADRC[TI]ITSARHNH[VF]NAPGR[IV]I(SEQ ID NO: 655); an amino acid sequence MOTIF 11 having at least 90%sequence identity to the amino acid sequence as represented by theformula[KV][VK][CA]RPPSPDM[MV]SAVAEHALWLNDVLLQVVQ[KN]ESQ[LM]QGT[AE]PYNECLAL LGR(SEQ ID NO: 656); an amino acid sequence MOTIF 12 having at least 90%sequence identity to the amino acid sequence as represented by theformula PTELT[VA]WPLGMDTV[AG]NLLIAQENAAL[VL]GLIQLGPSS (SEQ ID NO: 657);an amino acid sequence MOTIF 13 having at least 90% sequence identity tothe amino acid sequence as represented by the formulaRDQ[MT][HQ]MPGSVTVI[IV]LCRLLQFP[IT]DGSQA[TA]T (SEQ ID NO: 658); an aminoacid sequence MOTIF 14 having at least 90% sequence identity to theamino acid sequence as represented by the formulaTSIPVEVVTDP[SN]ILLGMQTTV[LH]IAEL (SEQ ID NO: 659); an amino acidsequence MOTIF 15 having at least 90% sequence identity to the aminoacid sequence as represented by the formulaEGLR[EQ]FQNRQVARA[VL]FAVLKAVA[MQ]I[AG] (SEQ ID NO: 660); an amino acidsequence MOTIF 16 having at least 90% sequence identity to the aminoacid sequence as represented by the formula W[TS]RVRIRHLEM[QH]F[AV]QEASG(SEQ ID NO: 661); an amino acid sequence MOTIF 17 having at least 90%sequence identity to the amino acid sequence as represented by theformula QISELQY[ED]IWVQG[LM][ML]RDIA (SEQ ID NO: 662); an amino acidsequence MOTIF 18 having at least 90% sequence identity to the aminoacid sequence as represented by the formulaTFTLGSGVTGITSMHGEPSLDPWNGVSLDSASPTAF (SEQ ID NO: 663); an amino acidsequence MOTIF 19 having at least 90% sequence identity to the aminoacid sequence as represented by the formula MDYSTLYRDLNQIS (SEQ ID NO:664); an amino acid sequence MOTIF 20 having at least 90% sequenceidentity to the amino acid sequence as represented by the formulaLRLPFM[QK]LHARVIEQN[VR]K[SE] (SEQ ID NO: 665); an amino acid sequenceMOTIF 21 having at least 90% sequence identity to the amino acidsequence as represented by the formula VDSLEQVG[QH][IL]V[GD]AP (SEQ IDNO: 666); an amino acid sequence MOTIF 22 having at least 90% sequenceidentity to the amino acid sequence as represented by the formula[IV][EQ][CA]VMK[IM]GRF[VG][SL]VV (SEQ ID NO: 667); an amino acidsequence MOTIF 23 having at least 90% sequence identity to the aminoacid sequence as represented by the formula TLTNEPSE[EQ]F (SEQ ID NO:668); and an amino acid sequence MOTIF 24 having at least 90% sequenceidentity to the amino acid sequence as represented by the formulaLPRQSRNISF (SEQ ID NO: 669).

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprises an amino acid sequence MOTIF selected from: anamino acid sequence MOTIF 1 as represented by an amino acid sequence ofthe formulaMP[DE]MP[ST][ED]ADWSIFVNE[IVL]EAVAEGMPTEVSEVP[AV]W[KR]AKCKN[MV]AALGREM[SC]I (SEQ ID NO: 670); an amino acid sequence MOTIF 2 as representedby an amino acid sequence of the formulaPQLQYRMYG[NS]LI[KRN]QMAQVAQNYD[QR][ED]FK[QR][FL][KR]LFI[IAVL]QNQI[LF]GSYLL[QE]QN[KR]AF (SEQ ID NO: 671); an amino acid sequence MOTIF 3 asrepresented by an amino acid sequence of the formulaN[TK]FMQMTPFT[RH]WRLRLSASA[SPKA]EN[AK][EG]LAFPTATA[PL]DSTT[EQ][IV][VA]ITFHVTAIR (SEQ ID NO: 672); an amino acid sequence MOTIF 4 as representedby an amino acid sequence of the formula[DN]FTSRHVVK[GD]IPV[SN]LLLDG[EG]DWEFEIPVQ[AG]GMSSFP (SEQ ID NO: 673); anamino acid sequence MOTIF 5 as represented by an amino acid sequence ofthe formulaIIHQP[SA]T[RQ][ST]G[IT][VI]YILLQGST[IV]FHDRRR[DE][EQ]V[ML]T[FP]QAA[DAV]PLN[FY][QH]YAYRLDTG(SEQ ID NO: 674); an amino acid sequence MOTIF 6 as represented by anamino acid sequence of the formulaS[HQ]ADRLAAIQP[AV][DN]LTN[HYF]LEMAT[HQ]MDMRTT[RS][MI]L[IL]GLLN[MI][LM]RIQNAAL[MR]YEY (SEQ ID NO: 675); an amino acid sequence MOTIF 7 asrepresented by an amino acid sequence of the formula[VL]D[RQ]VEFSEVMVIHRMYV[N]RL[SA]DL[ND]V[GA][EQ]L[PE]GA[EG][RK]VKR[VL]YV[FL]ADVVE(SEQ ID NO: 676); an amino acid sequence MOTIF 8 as represented by anamino acid sequence of the formulaA[DE]RELQMESFH[SA]AVISQ[RK]R[QGE]EL[ND][TD][AT][IF]AKM[DE]R[LM]SLQMEEE[NSD][RG]AMEQA[QR]KEM (SEQ ID NO: 677); an amino acid sequence MOTIF 9 asrepresented by an amino acid sequence of the formulaF[VL]TAGATAPGA[AV]ASAGQAV[SN]IAGQAAQ[AG]LRRVVEILE[GQ]LEAVMEVVAA[VI]K(SEQ ID NO: 678); an amino acid sequence MOTIF 10 as represented by anamino acid sequence of the formulaD[GD][MA][NK]WG[IT]Y[IV][YH][GA]E[KE]V[EQ][RVL]SPL[LYF][PN][SNG][NW][ASP][IY]L[AGV]V[WE]A[DQ]R[CS][TI]IT[SA]A[RFM]HN[HVT][VF][ND][AER]PG[RW][IV][IR] (SEQID NO: 679); an amino acid sequence MOTIF 11 as represented by an aminoacid sequence of the formula[KV][VK][CA][RGC][PHY]PSP[DE][MIL][MV]SAV[AG][EV]HA[LIN]WL[NS][DK]VLL[QR]VVQ[KN]ES[QH][LM]QGT[AE][PSA]YNECLALLGR (SEQ ID NO: 680); an amino acidsequence MOTIF 12 as represented by an amino acid sequence of theformula[PN]T[EQ]LT[VAT]WPL[GR]MDTV[AG][ND]LLI[AT][QH]E[NS]AAL[VLS]GL[ITMA]QLG[PQ][SP]S (SEQ ID NO: 681); an amino acid sequence MOTIF 13 as represented byan amino acid sequence of the formula[RLC][DLWK][QNPR][MTP][HQR][MIL]PGSVTVI[IV]LCRLLQFP[IT][DG]G[SR][QFR][AS][TAD][TW](SEQ ID NO: 682); an amino acid sequence MOTIF 14 as represented by anamino acid sequence of the formula[TA][SGV][IL]PV[ED]VVTDP[SN]IL[LM]GMQT[TS]V[LH]IAEL (SEQ ID NO: 683); anamino acid sequence MOTIF 15 as represented by an amino acid sequence ofthe formula EGLR[EQ]FQN[RE]QVA[RN]A[VL]FAVL[KS][AS]VA[MQ]I[AG] (SEQ IDNO: 684); an amino acid sequence MOTIF 16 as represented by an aminoacid sequence of the formula W[TS]RVRIRHLEM[QH]F[AV][QK]E[AS][SM][GN](SEQ ID NO: 685); an amino acid sequence MOTIF 17 as represented by anamino acid sequence of the formula Q[IM]S[EQ]LQY[ED]IWVQG[LM][ML]RD[IM]A(SEQ ID NO: 686); an amino acid sequence MOTIF 18 as represented by anamino acid sequence of the formula TFTLGSGVTGITSMHGEPSLDPWNGVSLDSASPTAF(SEQ ID NO: 663); an amino acid sequence MOTIF 19 as represented by anamino acid sequence of the formula [MLV]DY[SK][TSK]L[YF][RE]DLNQIS (SEQID NO: 687); an amino acid sequence MOTIF 20 as represented by an aminoacid sequence of the formulaL[RHQ]L[PT]FM[QK]LHA[RIT][VQL][IR]E[QER][NF][VR][KWS][SE] (SEQ ID NO:688); an amino acid sequence MOTIF 21 as represented by an amino acidsequence of the formula V[DN][SA]L[ED]QV[GS][QH][IL]V[GD]AP (SEQ ID NO:689); an amino acid sequence MOTIF 22 as represented by an amino acidsequence of the formula[IV][EQH][CAS][VA][MI]K[IM][GV][RP][FI][VG][SL]VV (SEQ ID NO: 690); anamino acid sequence MOTIF 23 as represented by an amino acid sequence ofthe formula TLTN[EQ]PSE[EQDH]F (SEQ ID NO: 691); and an amino acidsequence MOTIF 24 as represented by an amino acid sequence of theformula LP[RS]QS[RT]N[IV]SF (SEQ ID NO: 692).

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprises an amino acid sequence MOTIF selected from: anamino acid sequence MOTIF 1 having at least 90% sequence identity to theamino acid sequence as represented by the formulaMP[DE]MP[ST][ED]ADWSIFVNE[IVL]EAVAEGMPTEVSEVP[AV]W[KR]AKCKN[MV]AALGREM[SC]I (SEQ ID NO: 670); an amino acid sequence MOTIF 2 having at least90% sequence identity to the amino acid sequence as represented by theformulaPQLQYRMYG[NS]LI[KRN]QMAQVAQNYD[QR][ED]FK[QR][FL][KR]LFI[IAVL]QNQI[LF]GSYLL[QE]QN[KR]AF (SEQ ID NO: 671); an amino acid sequence MOTIF 3 having atleast 90% sequence identity to the amino acid sequence as represented bythe formulaN[TK]FMQMTPFT[RH]WRLRLSASA[SPKA]EN[AK][EG]LAFPTATA[PL]DSTT[EQ][IV][VA]ITFHVTAIR (SEQ ID NO: 672); an amino acid sequence MOTIF 4 having at least90% sequence identity to the amino acid sequence as represented by theformula [DN]FTSRHVVK[GD]IPV[SN]LLLDG[EG]DWEFEIPVQ[AG]GMSSFP (SEQ ID NO:673); an amino acid sequence MOTIF 5 having at least 90% sequenceidentity to the amino acid sequence as represented by the formulaIIHQP[SA]T[RQ][ST]G[IT][VI]YILLQGST[IV]FHDRRR[DE][EQ]V[ML]T[FP]QAA[DAV]PLN[FY][QH]YAYRLDTG(SEQ ID NO: 674); an amino acid sequence MOTIF 6 having at least 90%sequence identity to the amino acid sequence as represented by theformulaS[HQ]ADRLAAIQP[AV][DN]LTN[HYF]LEMAT[HQ]MDMRTT[RS][MI]L[IL]GLLN[MI][LM]RIQNAAL[MR]YEY (SEQ ID NO: 675); an amino acid sequence MOTIF 7 having atleast 90% sequence identity to the amino acid sequence as represented bythe formula[VL]D[RQ]VEFSEVMVIHRMYV[N]RL[SA]DL[ND]V[GA][EQ]L[PE]GA[EG][RK]VKR[VL]YV[FL]ADVVE(SEQ ID NO: 676); an amino acid sequence MOTIF 8 having at least 90%sequence identity to the amino acid sequence as represented by theformulaA[DE]RELQMESFH[SA]AVISQ[RK]R[QGE]EL[ND][TD][AT][IF]AKM[DE]R[LM]SLQMEEE[NSD][RG]AMEQA[QR]KEM (SEQ ID NO: 677); an amino acid sequence MOTIF 9having at least 90% sequence identity to the amino acid sequence asrepresented by the formulaF[VL]TAGATAPGA[AV]ASAGQAV[SN]IAGQAAQ[AG]LRRVVEILE[GQ]LEAVMEVVAA[VI]K(SEQ ID NO: 678); an amino acid sequence MOTIF 10 having at least 90%sequence identity to the amino acid sequence as represented by theformulaD[GD][MA][NK]WG[IT]Y[IV][YH][GA]E[KE]V[EQ][RVL]SPL[LYF][PN][SNG][NW][ASP][IY]L[AGV]V[WE]A[DQ]R[CS][TI]IT[SA]A[RFM]HN[HVT][VF][ND][AER]PG[RW][IV][IR] (SEQID NO: 679); an amino acid sequence MOTIF 11 having at least 90%sequence identity to the amino acid sequence as represented by theformula[KV][VK][CA][RGC][PHY]PSP[DE][MIL][MV]SAV[AG][EV]HA[LIN]WL[NS][DK]VLL[QR]VVQ[KN]ES[QH][LM]QGT[AE][PSA]YNECLALLGR (SEQ ID NO: 680); an amino acidsequence MOTIF 12 having at least 90% sequence identity to the aminoacid sequence as represented by the formula[PN]T[EQ]LT[VAT]WPL[GR]MDTV[AG][ND]LLI[AT][QH]E[NS]AAL[VLS]GL[ITMA]QLG[PQ][SP]S (SEQ ID NO: 681); an amino acid sequence MOTIF 13 having at least90% sequence identity to the amino acid sequence as represented by theformula[RLC][DLWK][QNPR][MTP][HQR][MIL]PGSVTVI[IV]LCRLLQFP[IT][DG]G[SR][QFR][AS][TAD][TW](SEQ ID NO: 682); an amino acid sequence MOTIF 14 having at least 90%sequence identity to the amino acid sequence as represented by theformula [TA][SGV][IL]PV[ED]VVTDP[SN]IL[LM]GMQT[TS]V[LH]IAEL (SEQ ID NO:683); an amino acid sequence MOTIF 15 having at least 90% sequenceidentity to the amino acid sequence as represented by the formulaEGLR[EQ]FQN[RE]QVA[RN]A[VL]FAVL[KS][AS]VA[MQ]I[AG](SEQ ID NO: 684); anamino acid sequence MOTIF 16 having at least 90% sequence identity tothe amino acid sequence as represented by the formulaW[TS]RVRIRHLEM[QH]F[AV][QK]E[AS][SM][GN] (SEQ ID NO: 685); an amino acidsequence MOTIF 17 having at least 90% sequence identity to the aminoacid sequence as represented by the formulaQ[IM]S[EQ]LQY[ED]IWVQG[LM][ML]RD[IM]A (SEQ ID NO: 686); an amino acidsequence MOTIF 18 having at least 90% sequence identity to the aminoacid sequence as represented by the formulaTFTLGSGVTGITSMHGEPSLDPWNGVSLDSASPTAF (SEQ ID NO: 663); an amino acidsequence MOTIF 19 having at least 90% sequence identity to the aminoacid sequence as represented by the formula[MLV]DY[SK][TSK]L[YF][RE]DLNQIS (SEQ ID NO: 687); an amino acid sequenceMOTIF 20 having at least 90% sequence identity to the amino acidsequence as represented by the formulaL[RHQ]L[PT]FM[QK]LHA[RIT][VQL][IR]E[QER][NF][VR][KWS][SE] (SEQ ID NO:688); an amino acid sequence MOTIF 21 having at least 90% sequenceidentity to the amino acid sequence as represented by the formulaV[DN][SA]L[ED]QV[GS][QH][IL]V[GD]AP (SEQ ID NO: 689); an amino acidsequence MOTIF 22 having at least 90% sequence identity to the aminoacid sequence as represented by the formula[IV][EQH][CAS][VA][MI]K[IM][GV][RP][FI][VG][SL]VV (SEQ ID NO: 690); anamino acid sequence MOTIF 23 having at least 90% sequence identity tothe amino acid sequence as represented by the formula TLTN[EQ]PSE[EQDH]F(SEQ ID NO: 691); and an amino acid sequence MOTIF 24 having at least90% sequence identity to the amino acid sequence as represented by theformula LP[RS]QS[RT]N[IV]SF (SEQ ID NO: 692).

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprises an amino acid sequence MOTIF selected from: anamino acid sequence MOTIF 1 as represented by an amino acid sequence ofthe formulaMP[DE]MP[ST][ED]ADWSIFVNE[IVL]EAVAEGMPTEVSEVP[AVIL]W[KR]AKCKN[MVIL]AALGREM[SCT]I (SEQ ID NO: 693); an amino acid sequence MOTIF 2 asrepresented by an amino acid sequence of the formulaPQLQYRMYG[NS]LI[KRNQ]QMAQVAQNYD[QRNK][ED]FK[QRNK][FL][KR]LFI[IAVL]QNQI[LFIV]GSYLL[QEND]QN[KR]AF (SEQ ID NO: 694); an amino acid sequence MOTIF 3as represented by an amino acid sequence of the formulaN[TKSR]FMQMTPFT[RHK]WRLRLSASA[SPKATR]EN[AKR][EG]LAFPTATA[PLIV]DSTT[EQND][IVL][VAIL]ITFHVTAIR (SEQ ID NO: 695); an amino acid sequence MOTIF 4as represented by an amino acid sequence of the formula[DNQE]FTSRHVVK[GDE]IPV[SNTQ]LLLDG[EGD]DWEFEIPVQ[AG]GMSSFP (SEQ ID NO:696); an amino acid sequence MOTIF 5 as represented by an amino acidsequence of the formulaIIHQP[SAT]T[RQKN][ST]G[ITLVS][VIL]YILLQGST[IVL]FHDRRR[DE][EQDN]V[MLIV]T[FP]QAA[DAVEIL]PLN[FY][QHN]YAYRLDTG (SEQ ID NO: 697); an amino acid sequenceMOTIF 6 as represented by an amino acid sequence of the formulaS[HQN]ADRLAAIQP[AVIL][DN]LTN[HYF]LEMAT[HQN]MDMRTT[RSKT][MILV]L[ILV]GLLN[MILV][LMIV]RIQNAAL[MRILVK]YEY (SEQ ID NO: 698); an amino acid sequenceMOTIF 7 as represented by an amino acid sequence of the formula[VLI]D[RQKN]VEFSEVMVIHRMYV[N]RL[SAT]DL[NDQE]V[GA][EQND]L[PED]GA[EGD][RK]VKR[VLI]YV[FLIV]ADVVE(SEQ ID NO: 699); an amino acid sequence MOTIF 8 as represented by anamino acid sequence of the formulaA[DE]RELQMESFH[SAT]AVISQ[RK]R[QGEND]EL[NDQE][TDSE][ATS][IFLV]AKM[DE]R[LMIV]SLQMEEE[NSDQET][RGK]AMEQA[QRNK]KEM (SEQ ID NO: 700); an amino acidsequence MOTIF 9 as represented by an amino acid sequence of the formulaF[VLI]TAGATAPGA[AVIL]ASAGQAV[SNTQ]IAGQAAQ[AG]LRRVVEILE[GQN]LEAVMEVVAA[VIL]K (SEQ ID NO: 701); an amino acid sequence MOTIF 10 as representedby an amino acid sequence of the formulaD[GDE][MA][NKQK]WG[ITLVS]Y[IVL][YH][GA]E[KERD]V[EQND][RVLKI]SPL[LYFIV][PNQ][SNGTQ][NWQ][ASPT][IYLV]L[AGVIL]V[WED]A[DQNE]R[CST][TISLV]IT[SAT]A[RFMK]HN[HVTILS][VFIL][NDQE][AERDK]PG[RWK][IVL][IRLVK] (SEQ ID NO: 702); an aminoacid sequence MOTIF 11 as represented by an amino acid sequence of theformula[KVRIL][VKRIL][CA][RGCK][PHY]PSP[DE][MILV][MVIL]SAV[AG][EVDIL]HA[LINVQ]WL[NSQT][DKER]VLL[QRNK]VVQ[KNRQ]ES[QHN][LMIV]QGT[AED][PSAT]YNECLALLGR (SEQ IDNO: 703); an amino acid sequence MOTIF 12 as represented by an aminoacid sequence of the formula[PNQ]T[EQDN]LT[VATILS]WPL[GRK]MDTV[AG][NDQE]LLI[ATS][QHN]E[NSQT]AAL[VLSIT]GL[ITMALVS]QLG[PQN][SPT]S(SEQ ID NO: 704); an amino acid sequence MOTIF 13 as represented by anamino acid sequence of the formula[RLCKIV][DLWKEIVR][QNPRK][MTP][HQR][MILV]PGSVTVI[IVL]LCRLLQFP[ITLVS][DGE]G[SRTK][QFRNK][AST][TADES][TWS](SEQ ID NO: 705); an amino acid sequence MOTIF 14 as represented by anamino acid sequence of the formula[TA][SGVTIL][ILV]PV[ED]VVTDP[SNTQ]IL[LMIV]GMQT[TS]V[LHIV]IAEL (SEQ IDNO: 706); an amino acid sequence MOTIF 15 as represented by an aminoacid sequence of the formulaEGLR[EQND]FQN[REKD]QVA[RNKQ]A[VLI]FAVL[KSRT][AST]VA[MQN]I[AG] (SEQ IDNO: 707); an amino acid sequence MOTIF 16 as represented by an aminoacid sequence of the formulaW[TS]RVRIRHLEM[QHN]F[AVIL][QKNR]E[AST][SMT][GNQ] (SEQ ID NO: 708); anamino acid sequence MOTIF 17 as represented by an amino acid sequence ofthe formula Q[IMLV]S[EQND]LQY[ED]IWVQG[LMIV][MLIV]RD[IMLV]A (SEQ ID NO:709); an amino acid sequence MOTIF 18 as represented by an amino acidsequence of the formula TFTLGSGVTGITSMHGEPSLDPWNGVSLDSASPTAF (SEQ ID NO:663); an amino acid sequence MOTIF 19 as represented by an amino acidsequence of the formula [MLVI]DY[SKTR][TSKR]L[YF][REKD]DLNQIS (SEQ IDNO: 710); an amino acid sequence MOTIF 20 as represented by an aminoacid sequence of the formulaL[RHQKN]L[PTS]FM[QKNR]LHA[RITKLVS][VQLIN][IRLVK]E[QERNDK][NFQ][VRILK][KWSRT][SETD] (SEQ ID NO: 711); an amino acid sequence MOTIF 21 asrepresented by an amino acid sequence of the formulaV[DNQE][SAT]L[ED]QV[GST][QHN][ILV]V[GDE]AP (SEQ ID NO: 712); an aminoacid sequence MOTIF 22 as represented by an amino acid sequence of theformula[IVL][EQHND][CAST][VAIL][MILV]K[IMLV][GVIL][RPK][FILV][VGIL][SLTIV]VV(SEQ ID NO: 713); an amino acid sequence MOTIF 23 as represented by anamino acid sequence of the formula TLTN[EQDN]PSE[EQDHN]F (SEQ ID NO:714); and an amino acid sequence MOTIF 24 as represented by an aminoacid sequence of the formula LP[RSKT]QS[RTKS]N[IVL]SF (SEQ ID NO: 715).

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprises an amino acid sequence MOTIF selected from: anamino acid sequence MOTIF 1 having at least 90% sequence identity to theamino acid sequence as represented by the formulaMP[DE]MP[ST][ED]ADWSIFVNE[IVL]EAVAEGMPTEVSEVP[AVIL]W[KR]AKCKN[MVIL]AALGREM[SCT]I (SEQ ID NO: 693); an amino acid sequence MOTIF 2 having atleast 90% sequence identity to the amino acid sequence as represented bythe formulaPQLQYRMYG[NS]LI[KRNQ]QMAQVAQNYD[QRNK][ED]FK[QRNK][FL][KR]LFI[IAVL]QNQI[LFIV]GSYLL[QEND]QN[KR]AF (SEQ ID NO: 694); an amino acid sequence MOTIF 3having at least 90% sequence identity to the amino acid sequence asrepresented by the formulaN[TKSR]FMQMTPFT[RHK]WRLRLSASA[SPKATR]EN[AKR][EG]LAFPTATA[PLIV]DSTT[EQND][IVL][VAIL]ITFHVTAIR (SEQ ID NO: 695); an amino acid sequence MOTIF 4having at least 90% sequence identity to the amino acid sequence asrepresented by the formula[DNQE]FTSRHVVK[GDE]IPV[SNTQ]LLLDG[EGD]DWEFEIPVQ[AG]GMSSFP (SEQ ID NO:696); an amino acid sequence MOTIF 5 having at least 90% sequenceidentity to the amino acid sequence as represented by the formulaIIHQP[SAT]T[RQKN][ST]G[ITLVS][VIL]YILLQGST[IVL]FHDRRR[DE][EQDN]V[MLIV]T[FP]QAA[DAVEIL]PLN[FY][QHN]YAYRLDTG (SEQ ID NO: 697); an amino acid sequenceMOTIF 6 having at least 90% sequence identity to the amino acid sequenceas represented by the formulaS[HQN]ADRLAAIQP[AVIL][DN]LTN[HYF]LEMAT[HQN]MDMRTT[RSKT][MILV]L[ILV]GLLN[MILV][LMIV]RIQNAAL[MRILVK]YEY (SEQ ID NO: 698); an amino acid sequenceMOTIF 7 having at least 90% sequence identity to the amino acid sequenceas represented by the formula[VLI]D[RQKN]VEFSEVMVIHRMYV[N]RL[SAT]DL[NDQE]V[GA][EQND]L[PED]GA[EGD][RK]VKR[VLI]YV[FLIV]ADVVE(SEQ ID NO: 699); an amino acid sequence MOTIF 8 having at least 90%sequence identity to the amino acid sequence as represented by theformulaA[DE]RELQMESFH[SAT]AVISQ[RK]R[QGEND]EL[NDQE][TDSE][ATS][IFLV]AKM[DE]R[LMIV]SLQMEEE[NSDQET][RGK]AMEQA[QRNK]KEM (SEQ ID NO: 700); an amino acidsequence MOTIF 9 having at least 90% sequence identity to the amino acidsequence as represented by the formulaF[VLI]TAGATAPGA[AVIL]ASAGQAV[SNTQ]IAGQAAQ[AG]LRRVVEILE[GQN]LEAVMEVVAA[VIL]K (SEQ ID NO: 701); an amino acid sequence MOTIF 10 having atleast 90% sequence identity to the amino acid sequence as represented bythe formulaD[GDE][MA][NKQK]WG[ITLVS]Y[IVL][YH][GA]E[KERD]V[EQND][RVLKI]SPL[LYFIV][PNQ][SNGTQ][NWQ][ASPT][IYLV]L[AGVIL]V[WED]A[DQNE]R[CST][TISLV]IT[SAT]A[RFMK]HN[HVTILS][VFIL][NDQE][AERDK]PG[RWK][IVL][IRLVK] (SEQ ID NO: 702); an aminoacid sequence MOTIF 11 having at least 90% sequence identity to theamino acid sequence as represented by the formula[KVRIL][VKRIL][CA][RGCK][PHY]PSP[DE][MILV][MVIL]SAV[AG][EVDIL]HA[LINVQ]WL[NSQT][DKER]VLL[QRNK]VVQ[KNRQ]ES[QHN][LMIV]QGT[AED][PSAT]YNECLALLGR (SEQ IDNO: 703); an amino acid sequence MOTIF 12 having at least 90% sequenceidentity to the amino acid sequence as represented by the formula[PNQ]T[EQDN]LT[VATILS]WPL[GRK]MDTV[AG][NDQE]LLI[ATS][QHN]E[NSQT]AAL[VLSIT]GL[ITMALVS]QLG[PQN][SPT]S(SEQ ID NO: 704); an amino acid sequence MOTIF 13 having at least 90%sequence identity to the amino acid sequence as represented by theformula[RLCKIV][DLWKEIVR][QNPRK][MTP][HQR][MILV]PGSVTVI[IVL]LCRLLQFP[ITLVS][DGE]G[SRTK][QFRNK][AST][TADES][TWS](SEQ ID NO: 705); an amino acid sequence MOTIF 14 having at least 90%sequence identity to the amino acid sequence as represented by theformula [TA][SGVTIL][ILV]PV[ED]VVTDP[SNTQ]IL[LMIV]GMQT[TS]V[LHIV]IAEL(SEQ ID NO: 706); an amino acid sequence MOTIF 15 having at least 90%sequence identity to the amino acid sequence as represented by theformula EGLR[EQND]FQN[REKD]QVA[RNKQ]A[VLI]FAVL[KSRT][AST]VA[MQN]I[AG](SEQ ID NO: 707); an amino acid sequence MOTIF 16 having at least 90%sequence identity to the amino acid sequence as represented by theformula W[TS]RVRIRHLEM[QHN]F[AVIL][QKNR]E[AST][SMT][GNQ] (SEQ ID NO:708); an amino acid sequence MOTIF 17 having at least 90% sequenceidentity to the amino acid sequence as represented by the formulaQ[IMLV]S[EQND]LQY[ED]IWVQG[LMIV][MLIV]RD[IMLV]A (SEQ ID NO: 709); anamino acid sequence MOTIF 18 having at least 90% sequence identity tothe amino acid sequence as represented by the formulaTFTLGSGVTGITSMHGEPSLDPWNGVSLDSASPTAF (SEQ ID NO: 663); an amino acidsequence MOTIF 19 having at least 90% sequence identity to the aminoacid sequence as represented by the formula[MLVI]DY[SKTR][TSKR]L[YF][REKD]DLNQIS (SEQ ID NO: 710); an amino acidsequence MOTIF 20 having at least 90% sequence identity to the aminoacid sequence as represented by the formulaL[RHQKN]L[PTS]FM[QKNR]LHA[RITKLVS][VQLIN][IRLVK]E[QERNDK][NFQ][VRILK][KWSRT][SETD] (SEQ ID NO: 711); an amino acid sequence MOTIF 21 having atleast 90% sequence identity to the amino acid sequence as represented bythe formula V[DNQE][SAT]L[ED]QV[GST][QHN][ILV]V[GDE]AP (SEQ ID NO: 712);an amino acid sequence MOTIF 22 having at least 90% sequence identity tothe amino acid sequence as represented by the formula[IVL][EQHND][CAST][VAIL][MILV]K[IMLV][GVIL][RPK][FILV][VGIL][SLTIV]VV(SEQ ID NO: 713); an amino acid sequence MOTIF 23 having at least 90%sequence identity to the amino acid sequence as represented by theformula TLTN[EQDN]PSE[EQDHN]F (SEQ ID NO: 714); and an amino acidsequence MOTIF 24 having at least 90% sequence identity to the aminoacid sequence as represented by the formula LP[RSKT]QS[RTKS]N[IVL]SF(SEQ ID NO: 715).

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprising, sequentially from the N-terminus to theC-terminus, an amino acid sequence MOTIF selected from: MOTIF 19 (SEQ IDNO: 664, SEQ ID NO: 687 or SEQ ID NO: 710), MOTIF 7 (SEQ ID NO: 652, SEQID NO: 676 or SEQ ID NO: 699), MOTIF 13 (SEQ ID NO: 658, SEQ ID NO: 682or SEQ ID NO: 705), MOTIF 20 (SEQ ID NO: 665, SEQ ID NO: 688 or SEQ IDNO: 711), MOTIF 10 (SEQ ID NO: 655, SEQ ID NO: 679 or SEQ ID NO: 702),MOTIF 18 (SEQ ID NO: 663), MOTIF 24 (SEQ ID NO: 669, SEQ ID NO: 692 orSEQ ID NO: 715), MOTIF 14 (SEQ ID NO: 659, SEQ ID NO: 683 or SEQ ID NO:706), MOTIF 11 (SEQ ID NO: 656, SEQ ID NO: 680 or SEQ ID NO: 703), MOTIF22 (SEQ ID NO: 667, SEQ ID NO: 690 or SEQ ID NO: 713), MOTIF 2 (SEQ IDNO: 647, SEQ ID NO: 671 or SEQ ID NO: 694), MOTIF 8 (SEQ ID NO: 653, SEQID NO: 677 or SEQ ID NO: 700), MOTIF 15 (SEQ ID NO: 660, SEQ ID NO: 684or SEQ ID NO: 707), MOTIF 9 (SEQ ID NO: 654, SEQ ID NO: 678 or SEQ IDNO: 701), MOTIF 21 (SEQ ID NO: 666, SEQ ID NO: 689 or SEQ ID NO: 712),MOTIF 1 (SEQ ID NO: 646, SEQ ID NO: 670 or SEQ ID NO: 693), MOTIF 17(SEQ ID NO: 662, SEQ ID NO: 686 or SEQ ID NO: 709), MOTIF 6 (SEQ ID NO:651, SEQ ID NO: 675 or SEQ ID NO: 698), MOTIF 12 (SEQ ID NO: 657, SEQ IDNO: 681 or SEQ ID NO: 704), MOTIF 4 (SEQ ID NO: 649, SEQ ID NO: 673 orSEQ ID NO: 696), MOTIF 16 (SEQ ID NO: 661, SEQ ID NO: 685 or SEQ ID NO:708), MOTIF 5 (SEQ ID NO: 650, SEQ ID NO: 674 or SEQ ID NO: 697), MOTIF23 (SEQ ID NO: 668, SEQ ID NO: 691 or SEQ ID NO: 714), and MOTIF 3 (SEQID NO: 648, SEQ ID NO: 672 or SEQ ID NO: 695).

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprising, sequentially from the N-terminus to theC-terminus, an amino acid sequence MOTIF selected from: MOTIF 19 (SEQ IDNO: 664, SEQ ID NO: 687 or SEQ ID NO: 710), MOTIF 7 (SEQ ID NO: 652, SEQID NO: 676 or SEQ ID NO: 699), MOTIF 13 (SEQ ID NO: 658, SEQ ID NO: 682or SEQ ID NO: 705), MOTIF 20 (SEQ ID NO: 665, SEQ ID NO: 688 or SEQ IDNO: 711), MOTIF 14 (SEQ ID NO: 659, SEQ ID NO: 683 or SEQ ID NO: 706),MOTIF 2 (SEQ ID NO: 647, SEQ ID NO: 671 or SEQ ID NO: 694), MOTIF 8 (SEQID NO: 653, SEQ ID NO: 677 or SEQ ID NO: 700), MOTIF 15 (SEQ ID NO: 660,SEQ ID NO: 684 or SEQ ID NO: 707), MOTIF 9 (SEQ ID NO: 654, SEQ ID NO:678 or SEQ ID NO: 701), MOTIF 21 (SEQ ID NO: 666, SEQ ID NO: 689 or SEQID NO: 712), MOTIF 1 (SEQ ID NO: 646, SEQ ID NO: 670 or SEQ ID NO: 693),MOTIF 17 (SEQ ID NO: 662, SEQ ID NO: 686 or SEQ ID NO: 709), MOTIF 6(SEQ ID NO: 651, SEQ ID NO: 675 or SEQ ID NO: 698), MOTIF 12 (SEQ ID NO:657, SEQ ID NO: 681 or SEQ ID NO: 704), MOTIF 4 (SEQ ID NO: 649, SEQ IDNO: 673 or SEQ ID NO: 696), MOTIF 16 (SEQ ID NO: 661, SEQ ID NO: 685 orSEQ ID NO: 708), MOTIF 5 (SEQ ID NO: 650, SEQ ID NO: 674 or SEQ ID NO:697), MOTIF 23 (SEQ ID NO: 668, SEQ ID NO: 691 or SEQ ID NO: 714), andMOTIF 3 (SEQ ID NO: 648, SEQ ID NO: 672 or SEQ ID NO: 695).

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprising, sequentially from the N-terminus to theC-terminus, the amino acid sequence motifs: MOTIF 19 (SEQ ID NO: 664,SEQ ID NO: 687 or SEQ ID NO: 710), MOTIF 7 (SEQ ID NO: 652, SEQ ID NO:676 or SEQ ID NO: 699), MOTIF 13 (SEQ ID NO: 658, SEQ ID NO: 682 or SEQID NO: 705), MOTIF 20 (SEQ ID NO: 665, SEQ ID NO: 688 or SEQ ID NO:711), MOTIF 10 (SEQ ID NO: 655, SEQ ID NO: 679 or SEQ ID NO: 702), MOTIF18 (SEQ ID NO: 663), MOTIF 24 (SEQ ID NO: 669, SEQ ID NO: 692 or SEQ IDNO: 715), MOTIF 14 (SEQ ID NO: 659, SEQ ID NO: 683 or SEQ ID NO: 706),MOTIF 11 (SEQ ID NO: 656, SEQ ID NO: 680 or SEQ ID NO: 703), MOTIF 22(SEQ ID NO: 667, SEQ ID NO: 690 or SEQ ID NO: 713), MOTIF 2 (SEQ ID NO:647, SEQ ID NO: 671 or SEQ ID NO: 694), MOTIF 8 (SEQ ID NO: 653, SEQ IDNO: 677 or SEQ ID NO: 700), MOTIF 15 (SEQ ID NO: 660, SEQ ID NO: 684 orSEQ ID NO: 707), MOTIF 9 (SEQ ID NO: 654, SEQ ID NO: 678 or SEQ ID NO:701), MOTIF 21 (SEQ ID NO: 666, SEQ ID NO: 689 or SEQ ID NO: 712), MOTIF1 (SEQ ID NO: 646, SEQ ID NO: 670 or SEQ ID NO: 693), MOTIF 17 (SEQ IDNO: 662, SEQ ID NO: 686 or SEQ ID NO: 709), MOTIF 6 (SEQ ID NO: 651, SEQID NO: 675 or SEQ ID NO: 698), MOTIF 12 (SEQ ID NO: 657, SEQ ID NO: 681or SEQ ID NO: 704), MOTIF 4 (SEQ ID NO: 649, SEQ ID NO: 673 or SEQ IDNO: 696), MOTIF 16 (SEQ ID NO: 661, SEQ ID NO: 685 or SEQ ID NO: 708),MOTIF 5 (SEQ ID NO: 650, SEQ ID NO: 674 or SEQ ID NO: 697), MOTIF 23(SEQ ID NO: 668, SEQ ID NO: 691 or SEQ ID NO: 714), and MOTIF 3 (SEQ IDNO: 648, SEQ ID NO: 672 or SEQ ID NO: 695).

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprising, sequentially from the N-terminus to theC-terminus, the amino acid sequence motifs: MOTIF 19 (SEQ ID NO: 664,SEQ ID NO: 687 or SEQ ID NO: 710), MOTIF 7 (SEQ ID NO: 652, SEQ ID NO:676 or SEQ ID NO: 699), MOTIF 13 (SEQ ID NO: 658, SEQ ID NO: 682 or SEQID NO: 705), MOTIF 20 (SEQ ID NO: 665, SEQ ID NO: 688 or SEQ ID NO:711), MOTIF 14 (SEQ ID NO: 659, SEQ ID NO: 683 or SEQ ID NO: 706), MOTIF2 (SEQ ID NO: 647, SEQ ID NO: 671 or SEQ ID NO: 694), MOTIF 8 (SEQ IDNO: 653, SEQ ID NO: 677 or SEQ ID NO: 700), MOTIF 15 (SEQ ID NO: 660,SEQ ID NO: 684 or SEQ ID NO: 707), MOTIF 9 (SEQ ID NO: 654, SEQ ID NO:678 or SEQ ID NO: 701), MOTIF 21 (SEQ ID NO: 666, SEQ ID NO: 689 or SEQID NO: 712), MOTIF 1 (SEQ ID NO: 646, SEQ ID NO: 670 or SEQ ID NO: 693),MOTIF 17 (SEQ ID NO: 662, SEQ ID NO: 686 or SEQ ID NO: 709), MOTIF 6(SEQ ID NO: 651, SEQ ID NO: 675 or SEQ ID NO: 698), MOTIF 12 (SEQ ID NO:657, SEQ ID NO: 681 or SEQ ID NO: 704), MOTIF 4 (SEQ ID NO: 649, SEQ IDNO: 673 or SEQ ID NO: 696), MOTIF 16 (SEQ ID NO: 661, SEQ ID NO: 685 orSEQ ID NO: 708), MOTIF 5 (SEQ ID NO: 650, SEQ ID NO: 674 or SEQ ID NO:697), MOTIF 23 (SEQ ID NO: 668, SEQ ID NO: 691 or SEQ ID NO: 714), andMOTIF 3 (SEQ ID NO: 648, SEQ ID NO: 672 or SEQ ID NO: 695).

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprising sequentially from the N-terminus to theC-terminus: a Region A of between about 200 to about 300 amino acids inlength comprising an amino acid sequence MOTIF of: MOTIF 19 (SEQ ID NO:664, SEQ ID NO: 687 or SEQ ID NO: 710), MOTIF 7 (SEQ ID NO: 652, SEQ IDNO: 676 or SEQ ID NO: 699), MOTIF 13 (SEQ ID NO: 658, SEQ ID NO: 682 orSEQ ID NO: 705), MOTIF 20 (SEQ ID NO: 665, SEQ ID NO: 688 or SEQ ID NO:711), MOTIF 10 (SEQ ID NO: 655, SEQ ID NO: 679 or SEQ ID NO: 702), MOTIF18 (SEQ ID NO: 663), MOTIF 24 (SEQ ID NO: 669, SEQ ID NO: 692 or SEQ IDNO: 715), and/or MOTIF 14 having a predominantly nonconserved secondarystructure; a Region B of between about 380 to about 465 amino acids inlength comprising an amino acid sequence MOTIF of MOTIF 22 (SEQ ID NO:667, SEQ ID NO: 690 or SEQ ID NO: 713), MOTIF 2 (SEQ ID NO: 647, SEQ IDNO: 671 or SEQ ID NO: 694), MOTIF 8 (SEQ ID NO: 653, SEQ ID NO: 677 orSEQ ID NO: 700), MOTIF 15 (SEQ ID NO: 660, SEQ ID NO: 684 or SEQ ID NO:707), MOTIF 9 (SEQ ID NO: 654, SEQ ID NO: 678 or SEQ ID NO: 701), MOTIF21 (SEQ ID NO: 666, SEQ ID NO: 689 or SEQ ID NO: 712), MOTIF 1 (SEQ IDNO: 646, SEQ ID NO: 670 or SEQ ID NO: 693), MOTIF 17 (SEQ ID NO: 662,SEQ ID NO: 686 or SEQ ID NO: 709), MOTIF 6 (SEQ ID NO: 651, SEQ ID NO:675 or SEQ ID NO: 698), and/or MOTIF 12 and having a predominately alphahelical structure; and a Region C of between about 150 to about 180amino acids in length comprising an amino acid sequence MOTIF of MOTIF16 (SEQ ID NO: 661, SEQ ID NO: 685 or SEQ ID NO: 708), MOTIF 5 (SEQ IDNO: 650, SEQ ID NO: 674 or SEQ ID NO: 697), MOTIF 23 (SEQ ID NO: 668,SEQ ID NO: 691 or SEQ ID NO: 714), and/or MOTIF 3 (SEQ ID NO: 648, SEQID NO: 672 or SEQ ID NO: 695), having a consensus secondary structurecomprising predominately beta strand structure.

In some embodiments the PtIP-83 polypeptide comprises an amino acidsequence MOTIF at the positions as shown in Table 2.

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprising sequentially from the N-terminus to theC-terminus: a Region A of between about 200 to about 300 amino acids inlength having a predominantly nonconserved secondary structure; a RegionB of between about 380 to about 465 amino acids in length having aconsensus secondary structure comprising 8 to 10 segments ofpredominately alpha helical structure; and a Region C of between about150 to about 180 amino acids in length having a consensus secondarystructure comprising 6 to 8 segments of predominately beta strandstructure. As used herein “predominantly nonconserved secondarystructure” means that the regions of secondary structure don'tconsistently align within the family of PtIP polypeptides. As usedherein “predominately alpha helical structure” means that secondarystructure prediction may have one or more gap of between 1 to 6 aminoacids of coil and/or beta strand structure intervening in the alphahelix structure. As used herein “predominately beta strand structure”means that secondary structure prediction may have one or more gap ofbetween 1 to 6 amino acids of coil and/or alpha helix structureintervening in the beta strand structure. In some embodiments thesecondary structure is generated by the PSIPRED, top ranked secondarystructure prediction method (Jones D T. (1999) J. Mol. Biol. 292:195-202).

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprising sequentially from the N-terminus to theC-terminus: a Region A of between about 200 to about 300 amino acids inlength having a predominantly nonconserved secondary structure; a RegionB of between about 380 to about 465 amino acids in length having aconsensus secondary structure comprising nine segments of predominatelyalpha helical structure; and a Region C of between about 150 to about180 amino acids in length having a consensus secondary structurecomprising seven segments of predominately beta strand structure.

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprising sequentially from the N-terminus to theC-terminus: a Region A of between about 200 to about 300 amino acids inlength having a predominantly nonconserved secondary structure, whereinthe Region A comprises a conserved beta strand 1 (β1a) of between about4 and about 12 amino acids in length within about amino acid residue 30to about amino acid residue 130 from the N-terminus of the PtIP-83polypeptide; a Region B of between about 380 to about 465 amino acids inlength having a consensus secondary structure comprising nine segmentsof predominately alpha helical structure; and a Region C of betweenabout 150 to about 180 amino acids in length having a consensussecondary structure comprising seven segments of predominately betastrand structure.

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprising sequentially from the N-terminus to theC-terminus: a Region A of between about 200 to about 300 amino acids inlength having a flexible consensus secondary structure, wherein theRegion A comprises a conserved beta strand 1 (β1a) of between about 4and about 12 amino acids in length, a coil of between about 3 and andabout 18 amino acids in length and a beta strand 2 (β1b) of betweenabout 4 and about 32 amino acids in length, within about amino acidresidue 50 to about amino acid residue 165 from the N-terminus of thePtIP-83 polypeptide; a Region B of between about 380 to about 465 aminoacids in length having a consensus secondary structure comprising ninesegments of predominately alpha helical structure; and a Region C ofbetween about 150 to about 180 amino acids in length having a consensussecondary structure comprising seven segments of predominately betastrand structure.

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprising sequentially from the N-terminus to theC-terminus: a Region A of between about 200 to about 300 amino acids inlength having a predominantly nonconserved secondary structure; a RegionB of between about 380 to about 465 amino acids in length having aconsensus secondary structure comprising sequentially: i) an alphahelix-1 of between about 10 and about 26 amino acids in length; ii) acoil-1 of between about 2 and about 8 amino acids in length flanked byalpha helix-1 and alpha helix-2; iii) an alpha helix-2 of between about15 and about 24 amino acids in length; iv) a coil-2 of between about 4and about 14 amino acids in length flanked by alpha helix-2 and alphahelix-3; v) an alpha helix 3 of between about 15 and about 27 aminoacids in length; vi) a coil-3 of between about 11 and about 13 aminoacids in length flanked by alpha helix-3 and alpha helix-4; vii) analpha helix-4 of about 24 180 amino acids in length; viii) a coil-4 ofbetween about 4 and about 5 amino acids in length flanked by alphahelix-4 and alpha helix-5; ix) an alpha helix-5 of between about 50 andabout 54 amino acids in length; x) a coil-5 of between about 11 andabout 17 amino acids in length flanked by alpha helix-5 and alphahelix-6; xi) an alpha helix-6 of between about 15 and about 16 aminoacids in length; xii) a coil-6 of between about 6 and about 9 aminoacids in length flanked by alpha helix-6 and alpha helix-7; xiii) analpha helix-7 of between about 49 and about 55 amino acids in length;xiv) a coil-7 of between about 3 and about 8 amino acids in lengthflanked by alpha helix-7 and alpha helix-8; xv) an alpha helix-8 ofbetween about 33 and about 36 amino acids in length; xvi) a coil-8 ofbetween about 14 and about 16 amino acids in length flanked by alphahelix-8 and alpha helix-9; xvii) an alpha helix-9 of between about 16and about 23 amino acids in length; xviii) a coil-9 of between about 21and about 28 amino acids in length flanked by alpha helix-9 and RegionC; and a Region C of between about 150 to about 180 amino acids inlength having a consensus secondary structure comprising seven segmentsof predominately beta strand structure.

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprising sequentially from the N-terminus to theC-terminus: a Region A of between about 200 to about 300 amino acids inlength having a predominantly nonconserved secondary structure; a RegionB of between about 380 to about 465 amino acids in length having aconsensus secondary structure comprising nine segments of predominatelyalpha helical structure; and a Region C of between about 150 to about180 amino acids in length having a consensus secondary structurecomprising sequentially: i) a beta strand-1 (β1) of between about 3amino acids and about 5 amino acids in length; ii) a coil of betweenabout 13 amino acids and about 17 amino acids in length; iii) a betastrand-2 (β2) of between about 7 amino acids and about 11 amino acids inlength; iv) a coil of between about 17 amino acids and about 23 aminoacids in length; v) a beta strand-3 (β3) of between about 5 amino acidsand about 7 amino acids in length; vi) a coil of between about 12 aminoacids and about 14 amino acids in length; vii) a beta strand-4 (β4) ofbetween about 5 amino acids and about 6 amino acids in length; viii) acoil of between about 2 amino acids and about 7 amino acids in length;ix) a beta strand-5 (β5) of between about 5 amino acids and about 7amino acids in length; x) a coil of between about 26 amino acids andabout 28 amino acids in length; xi) a beta strand-6 (β6) of betweenabout 5 amino acids and about 7 amino acids in length; xii) a coil ofbetween about 16 amino acids and about 20 amino acids in length; andxiii) a beta strand-1 (β7) of between about 13 amino acids and about 17amino acids in length.

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprising sequentially from the N-terminus to theC-terminus: a Region A of between about 200 to about 300 amino acids inlength having a predominantly nonconserved secondary structure; a RegionB of between about 380 to about 465 amino acids in length having aconsensus secondary structure comprising sequentially: i) an alphahelix-1 of between about 10 and about 26 amino acids in length; ii) acoil-1 of between about 2 and about 8 amino acids in length flanked byalpha helix-1 and alpha helix-2; iii) an alpha helix-2 of between about15 and about 24 amino acids in length; iv) a coil-2 of between about 4and about 14 amino acids in length flanked by alpha helix-2 and alphahelix-3; v) an alpha helix 3 of between about 15 and about 27 aminoacids in length; vi) a coil-3 of between about 11 and about 13 aminoacids in length flanked by alpha helix-3 and alpha helix-4; vii) analpha helix-4 of about 24 180 amino acids in length; viii) a coil-4 ofbetween about 4 and about 5 amino acids in length flanked by alphahelix-4 and alpha helix-5; ix) an alpha helix-5 of between about 50 andabout 54 amino acids in length; x) a coil-5 of between about 11 andabout 17 amino acids in length flanked by alpha helix-5 and alphahelix-6; xi) an alpha helix-6 of between about 15 and about 16 aminoacids in length; xii) a coil-6 of between about 6 and about 9 aminoacids in length flanked by alpha helix-6 and alpha helix-7; xiii) analpha helix-7 of between about 49 and about 55 amino acids in length;xiv) a coil-7 of between about 3 and about 8 amino acids in lengthflanked by alpha helix-7 and alpha helix-8; xv) an alpha helix-8 ofbetween about 33 and about 36 amino acids in length; xvi) a coil-8 ofbetween about 14 and about 16 amino acids in length flanked by alphahelix-8 and alpha helix-9; xvii) an alpha helix-9 of between about 16and about 23 amino acids in length; xviii) a coil-9 of between about 21and about 28 amino acids in length flanked by alpha helix-9 and RegionC; and a Region C of between about 150 to about 180 amino acids inlength having a consensus secondary structure comprising sequentially:i) a beta strand-1 (β1) of between about 3 amino acids and about 5 aminoacids in length; ii) a coil of between about 13 amino acids and about 17amino acids in length; iii) a beta strand-2 (β2) of between about 7amino acids and about 11 amino acids in length; iv) a coil of betweenabout 17 amino acids and about 23 amino acids in length; v) a betastrand-3 (β3) of between about 5 amino acids and about 7 amino acids inlength; vi) a coil of between about 12 amino acids and about 14 aminoacids in length; vii) a beta strand-4 (β4) of between about 5 aminoacids and about 6 amino acids in length; viii) a coil of between about 2amino acids and about 7 amino acids in length; ix) a beta strand-5 (β5)of between about 5 amino acids and about 7 amino acids in length; x) acoil of between about 26 amino acids and about 28 amino acids in length;xi) a beta strand-6 (β6) of between about 5 amino acids and about 7amino acids in length; xii) a coil of between about 16 amino acids andabout 20 amino acids in length; and xiii) a beta strand-1 (β7) ofbetween about 13 amino acids and about 17 amino acids in length.

In some embodiments the nucleic acid molecule encodes a PtIP-83polypeptide comprising sequentially from the N-terminus to theC-terminus: a Region A of between about 200 to about 300 amino acids inlength having a flexible consensus secondary structure, wherein theRegion A comprises a conserved beta strand 1 (β1a) of between about 4and about 12 amino acids in length within about amino acid residue 30 toabout amino acid residue 130 from the N-terminus of the PtIP-83polypeptide; a Region B of between about 380 to about 465 amino acids inlength having a consensus secondary structure comprising sequentially:i) an alpha helix-1 of between about 10 and about 26 amino acids inlength; ii) a coil-1 of between about 2 and about 8 amino acids inlength flanked by alpha helix-1 and alpha helix-2; iii) an alpha helix-2of between about 15 and about 24 amino acids in length; iv) a coil-2 ofbetween about 4 and about 14 amino acids in length flanked by alphahelix-2 and alpha helix-3; v) an alpha helix 3 of between about 15 andabout 27 amino acids in length; vi) a coil-3 of between about 11 andabout 13 amino acids in length flanked by alpha helix-3 and alphahelix-4; vii) an alpha helix-4 of about 24 180 amino acids in length;viii) a coil-4 of between about 4 and about 5 amino acids in lengthflanked by alpha helix-4 and alpha helix-5; ix) an alpha helix-5 ofbetween about 50 and about 54 amino acids in length; x) a coil-5 ofbetween about 11 and about 17 amino acids in length flanked by alphahelix-5 and alpha helix-6; xi) an alpha helix-6 of between about 15 andabout 16 amino acids in length; xii) a coil-6 of between about 6 andabout 9 amino acids in length flanked by alpha helix-6 and alphahelix-7; xiii) an alpha helix-7 of between about 49 and about 55 aminoacids in length; xiv) a coil-7 of between about 3 and about 8 aminoacids in length flanked by alpha helix-7 and alpha helix-8; xv) an alphahelix-8 of between about 33 and about 36 amino acids in length; xvi) acoil-8 of between about 14 and about 16 amino acids in length flanked byalpha helix-8 and alpha helix-9; xvii) an alpha helix-9 of between about16 and about 23 amino acids in length; xviii) a coil-9 of between about21 and about 28 amino acids in length flanked by alpha helix-9 andRegion C; and a Region C of between about 150 to about 180 amino acidsin length having a consensus secondary comprising sequentially: i) abeta strand-1 (β1) of between about 3 amino acids and about 5 aminoacids in length; ii) a coil of between about 13 amino acids and about 17amino acids in length; iii) a beta strand-2 (β2) of between about 7amino acids and about 11 amino acids in length; iv) a coil of betweenabout 17 amino acids and about 23 amino acids in length; v) a betastrand-3 (β3) of between about 5 amino acids and about 7 amino acids inlength; vi) a coil of between about 12 amino acids and about 14 aminoacids in length; vii) a beta strand-4 (β4) of between about 5 aminoacids and about 6 amino acids in length; viii) a coil of between about 2amino acids and about 7 amino acids in length; ix) a beta strand-5 (β5)of between about 5 amino acids and about 7 amino acids in length; x) acoil of between about 26 amino acids and about 28 amino acids in length;xi) a beta strand-6 (β6) of between about 5 amino acids and about 7amino acids in length; xii) a coil of between about 16 amino acids andabout 20 amino acids in length; and xiii) a beta strand-1 (β7) ofbetween about 13 amino acids and about 17 amino acids in length.

Also provided are nucleic acid molecules that encode transcriptionand/or translation products that are subsequently spliced to ultimatelyproduce functional PtIP-83 polypeptides. Splicing can be accomplished invitro or in vivo, and can involve cis- or trans-splicing. The substratefor splicing can be polynucleotides (e.g., RNA transcripts) orpolypeptides. An example of cis-splicing of a polynucleotide is where anintron inserted into a coding sequence is removed and the two flankingexon regions are spliced to generate a PtIP-83 polypeptide encodingsequence. An example of trans splicing would be where a polynucleotideis encrypted by separating the coding sequence into two or morefragments that can be separately transcribed and then spliced to formthe full-length pesticidal encoding sequence. The use of a splicingenhancer sequence, which can be introduced into a construct, canfacilitate splicing either in cis or trans-splicing of polypeptides(U.S. Pat. Nos. 6,365,377 and 6,531,316). Thus, in some embodiments thepolynucleotides do not directly encode a full-length PtIP-83polypeptide, but rather encode a fragment or fragments of a PtIP-83polypeptide. These polynucleotides can be used to express a functionalPtIP-83 polypeptide through a mechanism involving splicing, wheresplicing can occur at the level of polynucleotide (e.g., intron/exon)and/or polypeptide (e.g., intein/extein). This can be useful, forexample, in controlling expression of pesticidal activity, since afunctional pesticidal polypeptide will only be expressed if all requiredfragments are expressed in an environment that permits splicingprocesses to generate functional product. In another example,introduction of one or more insertion sequences into a polynucleotidecan facilitate recombination with a low homology polynucleotide; use ofan intron or intein for the insertion sequence facilitates the removalof the intervening sequence, thereby restoring function of the encodedvariant.

Nucleic acid molecules that are fragments of these nucleic acidsequences encoding PtIP-83 polypeptides are also encompassed by theembodiments. “Fragment” as used herein refers to a portion of thenucleic acid sequence encoding a PtIP-83 polypeptide. A fragment of anucleic acid sequence may encode a biologically active portion of aPtIP-83 polypeptide or it may be a fragment that can be used as ahybridization probe or PCR primer using methods disclosed below. Nucleicacid molecules that are fragments of a nucleic acid sequence encoding aPtIP-83 polypeptide comprise at least about 150, 180, 210, 240, 270,300, 330 or 360, contiguous nucleotides or up to the number ofnucleotides present in a full-length nucleic acid sequence encoding aPtIP-83 polypeptide disclosed herein, depending upon the intended use.“Contiguous nucleotides” is used herein to refer to nucleotide residuesthat are immediately adjacent to one another. Fragments of the nucleicacid sequences of the embodiments will encode protein fragments thatretain the biological activity of the PtIP-83 polypeptide and, hence,retain insecticidal activity. “Retains insecticidal activity” is usedherein to refer to a polypeptide having at least about 10%, at leastabout 30%, at least about 50%, at least about 70%, 80%, 90%, 95% orhigher of the insecticidal activity of the full-length PtIP-83Aapolypeptide (SEQ ID NO: 1). In some embodiments, the insecticidalactivity is Lepidoptera activity. In one embodiment, the insecticidalactivity is against a Coleopteran species. In some embodiments, theinsecticidal activity is against one or more insect pests of the cornrootworm complex: western corn rootworm, Diabrotica virgifera; northerncorn rootworm, D. barberi: Southern corn rootworm or spotted cucumberbeetle; Diabrotica undecimpunctata howardi, and the Mexican cornrootworm, D. virgifera zeae. In one embodiment, the insecticidalactivity is against a Diabrotica species.

In some embodiments a fragment of a nucleic acid sequence encoding aPtIP-83 polypeptide encoding a biologically active portion of a proteinwill encode at least about 15, 20, 30, 50, 75, 100, 125, contiguousamino acids or up to the total number of amino acids present in afull-length PtIP-83 polypeptide of the embodiments. In some embodiments,the fragment is an N-terminal and/or a C-terminal truncation of at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or more aminoacids from the N-terminus and/or C-terminus relative to SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23 or SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755, SEQID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID NO:760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID NO: 764, SEQID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768 or SEQ ID NO:769 variants thereof, e.g., by proteolysis, insertion of a start codon,deletion of the codons encoding the deleted amino acids with theconcomitant insertion of a stop codon or by insertion of a stop codon inthe coding sequence. In some embodiments, the fragments encompassedherein result from the removal of the N-terminal 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore amino acids from the N-terminus relative to SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ IDNO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQID NO: 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO:756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID NO:765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768, SEQ ID NO: 769 orvariants thereof, e.g., by proteolysis or by insertion of a start codonin the coding sequence. In some embodiments, the fragments encompassedherein result from the removal of the N-terminal 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14 amino acids relative to SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ IDNO: 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756,SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ IDNO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765,SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768, SEQ ID NO: 769 orvariants thereof, e.g., by proteolysis or by insertion of a start codonin the coding sequence.

In some embodiments the PtIP-83 polypeptide is encoded by a nucleic acidsequence sufficiently homologous to the nucleic acid sequence of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQID NO: 22, SEQ ID NO: 24, SEQ ID NO: 717, SEQ ID NO: 738, SEQ ID NO:739, SEQ ID NO: 740, SEQ ID NO: 741, SEQ ID NO: 742, SEQ ID NO: 743, SEQID NO: 744, SEQ ID NO: 745, SEQ ID NO: 746, SEQ ID NO: 747, SEQ ID NO:748, SEQ ID NO: 749, SEQ ID NO: 750, SEQ ID NO: 751, SEQ ID NO: 752 orSEQ ID NO: 753. “Sufficiently homologous” is used herein to refer to anamino acid or nucleic acid sequence that has at least about 50%, 55%,60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequencehomology compared to a reference sequence using one of the alignmentprograms described herein using standard parameters. One of skill in theart will recognize that these values can be appropriately adjusted todetermine corresponding homology of proteins encoded by two nucleic acidsequences by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning, and the like.

In some embodiments the sequence homology is against the full lengthsequence of the polynucleotide encoding a PtIP-83 polypeptide or againstthe full length sequence of a PtIP-83 polypeptide.

In some embodiments the nucleic acid encoding a PtIP-83 polypeptide isselected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ IDNO: 29, any one of SEQ ID NO: 172-235, any one of SEQ ID NO: 300-333,any one of SEQ ID NO: 368-397, any one of SEQ ID NO: 428-517, SEQ ID NO:717, any one of SEQ ID NO: 718-727, and any one of SEQ ID NO: 738-753.

In some embodiments the nucleic acid encodes a PtIP-83 polypeptidehaving at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence identity compared to SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ IDNO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQID NO: 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO:756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID NO:765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768 or SEQ ID NO: 769.In some embodiments the sequence identity is calculated using ClustalWalgorithm in the ALIGNX® module of the Vector NTI® Program Suite(Invitrogen Corporation, Carlsbad, Calif.) with all default parameters.In some embodiments the sequence identity is across the entire length ofpolypeptide calculated using ClustalW algorithm in the ALIGNX module ofthe Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, Calif.)with all default parameters.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. In another embodiment, the comparison is across theentirety of the reference sequence (e.g., across the entirety of SEQ IDNO: 1). The percent identity between two sequences can be determinedusing techniques similar to those described below, with or withoutallowing gaps. In calculating percent identity, typically exact matchesare counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul, (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul, (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTNand BLASTX programs of Altschul, et al., (1990) J. Mol. Biol. 215:403.BLAST nucleotide searches can be performed with the BLASTN program,score=100, wordlength=12, to obtain nucleic acid sequences homologous topesticidal nucleic acid molecules of the embodiments. BLAST proteinsearches can be performed with the BLASTX program, score=50,wordlength=3, to obtain amino acid sequences homologous to pesticidalprotein molecules of the embodiments. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul, et al., (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationships between molecules. See, Altschul, et al.,(1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g.,BLASTX and BLASTN) can be used. Alignment may also be performed manuallyby inspection.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the ClustalW algorithm (Higgins, et al.,(1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences andaligns the entirety of the amino acid or DNA sequence, and thus canprovide data about the sequence conservation of the entire amino acidsequence. The ClustalW algorithm is used in several commerciallyavailable DNA/amino acid analysis software packages, such as the ALIGNX®module of the Vector NTI® Program Suite (Invitrogen Corporation,Carlsbad, Calif.). After alignment of amino acid sequences withClustalW, the percent amino acid identity can be assessed. Anon-limiting example of a software program useful for analysis ofClustalW alignments is GENEDOC™. GENEDOC™ (Karl Nicholas) allowsassessment of amino acid (or DNA) similarity and identity betweenmultiple proteins. Another non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller, (1988) CABIOS 4:11-17. Such an algorithm isincorporated into the ALIGN program (version 2.0), which is part of theGCG Wisconsin Genetics Software Package, Version 10 (available fromAccelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA). Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Needleman and Wunsch,(1970) J. Mol. Biol. 48(3):443-453, used GAP Version 10 software todetermine sequence identity or similarity using the following defaultparameters: % identity and % similarity for a nucleic acid sequenceusing GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmpiiscoring matrix; % identity or % similarity for an amino acid sequenceusing GAP weight of 8 and length weight of 2, and the BLOSUM62 scoringprogram. Equivalent programs may also be used. “Equivalent program” isused herein to refer to any sequence comparison program that, for anytwo sequences in question, generates an alignment having identicalnucleotide residue matches and an identical percent sequence identitywhen compared to the corresponding alignment generated by GAP Version10.

The embodiments also encompass nucleic acid molecules encoding PtIP-83polypeptide variants. “Variants” of the PtIP-83 polypeptide encodingnucleic acid sequences include those sequences that encode the PtIP-83polypeptides disclosed herein but that differ conservatively because ofthe degeneracy of the genetic code as well as those that aresufficiently identical as discussed above. Naturally occurring allelicvariants can be identified with the use of well-known molecular biologytechniques, such as polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant nucleic acid sequences alsoinclude synthetically derived nucleic acid sequences that have beengenerated, for example, by using site-directed mutagenesis but whichstill encode the PtIP-83 polypeptides disclosed as discussed below.

The present disclosure provides isolated or recombinant polynucleotidesthat encode any of the PtIP-83 polypeptides disclosed herein. Thosehaving ordinary skill in the art will readily appreciate that due to thedegeneracy of the genetic code, a multitude of nucleotide sequencesencoding PtIP-83 polypeptides of the present disclosure exist. Table 1is a codon table that provides the synonymous codons for each aminoacid. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU allencode the amino acid arginine. Thus, at every position in the nucleicacids of the disclosure where an arginine is specified by a codon, thecodon can be altered to any of the corresponding codons described abovewithout altering the encoded polypeptide. It is understood that U in anRNA sequence corresponds to T in a DNA sequence.

TABLE 1 Alanine Ala A GCA GCC GCG GC Cystine Cys UGC UGU Aspartic acidAsp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUUGlycine Gly G GGA GGC GGG GGU Histidine His CAC CAU Isoleucine II IAUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG C UMethionine Me M AUG  Asparagine Asn N AAC AAU Proline Pro PCCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg RAGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCC ThreonineThr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W TGG Tyrosine Tyr Y UAC UAU

The skilled artisan will further appreciate that changes can beintroduced by mutation of the nucleic acid sequences thereby leading tochanges in the amino acid sequence of the encoded PtIP-83 polypeptides,without altering the biological activity of the proteins. Thus, variantnucleic acid molecules can be created by introducing one or morenucleotide substitutions, additions and/or deletions into thecorresponding nucleic acid sequence disclosed herein, such that one ormore amino acid substitutions, additions or deletions are introducedinto the encoded protein. Mutations can be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleic acid sequences are also encompassed bythe present disclosure.

Alternatively, variant nucleic acid sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forability to confer pesticidal activity to identify mutants that retainactivity. Following mutagenesis, the encoded protein can be expressedrecombinantly, and the activity of the protein can be determined usingstandard assay techniques.

The polynucleotides of the disclosure and fragments thereof areoptionally used as substrates for a variety of recombination andrecursive recombination reactions, in addition to standard cloningmethods as set forth in, e.g., Ausubel, Berger and Sambrook, i.e., toproduce additional pesticidal polypeptide homologues and fragmentsthereof with desired properties. A variety of such reactions are known,including those developed by the inventors and their co-workers. Methodsfor producing a variant of any nucleic acid listed herein comprisingrecursively recombining such polynucleotide with a second (or more)polynucleotide, thus forming a library of variant polynucleotides arealso embodiments of the disclosure, as are the libraries produced, thecells comprising the libraries and any recombinant polynucleotideproduces by such methods. Additionally, such methods optionally compriseselecting a variant polynucleotide from such libraries based onpesticidal activity, as is wherein such recursive recombination is donein vitro or in vivo.

A variety of diversity generating protocols, including nucleic acidrecursive recombination protocols are available and fully described inthe art. The procedures can be used separately, and/or in combination toproduce one or more variants of a nucleic acid or set of nucleic acids,as well as variants of encoded proteins. Individually and collectively,these procedures provide robust, widely applicable ways of generatingdiversified nucleic acids and sets of nucleic acids (including, e.g.,nucleic acid libraries) useful, e.g., for the engineering or rapidevolution of nucleic acids, proteins, pathways, cells and/or organismswith new and/or improved characteristics.

While distinctions and classifications are made in the course of theensuing discussion for clarity, it will be appreciated that thetechniques are often not mutually exclusive. Indeed, the various methodscan be used singly or in combination, in parallel or in series, toaccess diverse sequence variants.

The result of any of the diversity generating procedures describedherein can be the generation of one or more nucleic acids, which can beselected or screened for nucleic acids with or which confer desirableproperties or that encode proteins with or which confer desirableproperties. Following diversification by one or more of the methodsherein or otherwise available to one of skill, any nucleic acids thatare produced can be selected for a desired activity or property, e.g.pesticidal activity or, such activity at a desired pH, etc. This caninclude identifying any activity that can be detected, for example, inan automated or automatable format, by any of the assays in the art,see, e.g., discussion of screening of insecticidal activity, infra. Avariety of related (or even unrelated) properties can be evaluated, inserial or in parallel, at the discretion of the practitioner.

Descriptions of a variety of diversity generating procedures forgenerating modified nucleic acid sequences, e.g., those coding forpolypeptides having pesticidal activity or fragments thereof, are foundin the following publications and the references cited therein: Soong,et al., (2000) Nat Genet 25(4):436-439; Stemmer, et al., (1999) TumorTargeting 4:1-4; Ness, et al., (1999) Nat Biotechnol 17:893-896; Chang,et al., (1999) Nat Biotechnol 17:793-797; Minshull and Stemmer, (1999)Curr Opin Chem Biol 3:284-290; Christians, et al., (1999) Nat Biotechnol17:259-264; Crameri, et al., (1998) Nature 391:288-291; Crameri, et al.,(1997) Nat Biotechnol 15:436-438; Zhang, et al., (1997) PNAS USA94:4504-4509; Patten, et al., (1997) Curr Opin Biotechnol 8:724-733;Crameri, et al., (1996) Nat Med 2:100-103; Crameri, et al., (1996) NatBiotechnol 14:315-319; Gates, et al., (1996) J Mol Biol 255:373-386;Stemmer, (1996) “Sexual PCR and Assembly PCR” In: The Encyclopedia ofMolecular Biology. VCH Publishers, New York. pp. 447-457; Crameri andStemmer, (1995) BioTechniques 18:194-195; Stemmer, et al., (1995) Gene,164:49-53; Stemmer, (1995) Science 270: 1510; Stemmer, (1995)Bio/Technology 13:549-553; Stemmer, (1994) Nature 370:389-391 andStemmer, (1994) PNAS USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling, et al., (1997) Anal Biochem254(2):157-178; Dale, et al., (1996) Methods Mol Biol 57:369-374; Smith,(1985) Ann Rev Genet 19:423-462; Botstein and Shortle, (1985) Science229:1193-1201; Carter, (1986) Biochem J 237:1-7 and Kunkel, (1987) “Theefficiency of oligonucleotide directed mutagenesis” in Nucleic Acids &Molecular Biology (Eckstein and Lilley, eds., Springer Verlag, Berlin));mutagenesis using uracil containing templates (Kunkel, (1985) PNAS USA82:488-492; Kunkel, et al., (1987) Methods Enzymol 154:367-382 and Bass,et al., (1988) Science 242:240-245); oligonucleotide-directedmutagenesis (Zoller and Smith, (1983) Methods Enzymol 100:468-500;Zoller and Smith, (1987) Methods Enzymol 154:329-350 (1987); Zoller andSmith, (1982) Nucleic Acids Res 10:6487-6500), phosphorothioate-modifiedDNA mutagenesis (Taylor, et al., (1985) Nucl Acids Res 13:8749-8764;Taylor, et al., (1985) Nucl Acids Res 13:8765-8787 (1985); Nakamaye andEckstein, (1986) Nucl Acids Res 14:9679-9698; Sayers, et al., (1988)Nucl Acids Res 16:791-802 and Sayers, et al., (1988) Nucl Acids Res16:803-814); mutagenesis using gapped duplex DNA (Kramer, et al., (1984)Nucl Acids Res 12:9441-9456; Kramer and Fritz, (1987) Methods Enzymol154:350-367; Kramer, et al., (1988) Nucl Acids Res 16:7207 and Fritz, etal., (1988) Nucl Acids Res 16:6987-6999).

Additional suitable methods include point mismatch repair (Kramer, etal., (1984) Cell 38:879-887), mutagenesis using repair-deficient hoststrains (Carter, et al., (1985) Nucl Acids Res 13:4431-4443 and Carter,(1987) Methods in Enzymol 154:382-403), deletion mutagenesis(Eghtedarzadeh and Henikoff, (1986) Nucl Acids Res 14:5115),restriction-selection and restriction-purification (Wells, et al.,(1986) Phil Trans R Soc Lond A 317:415-423), mutagenesis by total genesynthesis (Nambiar, et al., (1984) Science 223:1299-1301; Sakamar andKhorana, (1988) Nucl Acids Res 14:6361-6372; Wells, et al., (1985) Gene34:315-323 and Grundström, et al., (1985) Nucl Acids Res 13:3305-3316),double-strand break repair (Mandecki, (1986) PNAS USA, 83:7177-7181 andArnold, (1993) Curr Opin Biotech 4:450-455). Additional details on manyof the above methods can be found in Methods Enzymol Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Additional details regarding various diversity generating methods can befound in the following US Patents, PCT Publications and Applications andEPO publications: U.S. Pat. Nos. 5,723,323, 5,763,192, 5,814,476,5,817,483, 5,824,514, 5,976,862, 5,605,793, 5,811,238, 5,830,721,5,834,252, 5,837,458, WO 1995/22625, WO 1996/33207, WO 1997/20078, WO1997/35966, WO 1999/41402, WO 1999/41383, WO 1999/41369, WO 1999/41368,EP 752008, EP 0932670, WO 1999/23107, WO 1999/21979, WO 1998/31837, WO1998/27230, WO 1998/27230, WO 2000/00632, WO 2000/09679, WO 1998/42832,WO 1999/29902, WO 1998/41653, WO 1998/41622, WO 1998/42727, WO2000/18906, WO 2000/04190, WO 2000/42561, WO 2000/42559, WO 2000/42560,WO 2001/23401 and PCT/US01/06775.

The nucleotide sequences of the embodiments can also be used to isolatecorresponding sequences from ferns or other primitive plant,particularly a Asplenium, Polypodium, Adiantum, Platycerium,Nephrolepis, Ophioglossum, Colysis, Bolbitis, Blechnum, Selaginella,Lycopodium, and Huperzia species. In this manner, methods such as PCR,hybridization, and the like can be used to identify such sequences basedon their sequence homology to the sequences set forth herein. Sequencesthat are selected based on their sequence identity to the entiresequences set forth herein or to fragments thereof are encompassed bythe embodiments. Such sequences include sequences that are orthologs ofthe disclosed sequences. The term “orthologs” refers to genes derivedfrom a common ancestral gene and which are found in different species asa result of speculation. Genes found in different species are consideredorthologs when their nucleotide sequences and/or their encoded proteinsequences share substantial identity as defined elsewhere herein.Functions of orthologs are often highly conserved among species.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook, et al., (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.), hereinafter “Sambrook”. See also, Innis, et al., eds.(1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

To identify potential PtIP-83 polypeptides from fern or mosscollections, the fern or moss cell lysates can be screened withantibodies generated against a PtIP-83 polypeptides and/or PtIP-83polypeptides using Western blotting and/or ELISA methods. This type ofassays can be performed in a high throughput fashion. Positive samplescan be further analyzed by various techniques such as antibody basedprotein purification and identification. Methods of generatingantibodies are well known in the art as discussed infra.

Alternatively, mass spectrometry based protein identification method canbe used to identify homologs of PtIP-83 polypeptides using protocols inthe literatures (Scott Patterson, (1998), 10.22, 1-24, Current Protocolin Molecular Biology published by John Wiley & Son Inc). Specifically,LC-MS/MS based protein identification method is used to associate the MSdata of given cell lysate or desired molecular weight enriched samples(excised from SDS-PAGE gel of relevant molecular weight bands to PtIP-83polypeptides) with sequence information of PtIP-83 polypeptides SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO:755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID NO:764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768 orSEQ ID NO: 769 and their homologs. Any match in peptide sequencesindicates the potential of having the homologous proteins in thesamples. Additional techniques (protein purification and molecularbiology) can be used to isolate the protein and identify the sequencesof the homologs.

In hybridization methods, all or part of the pesticidal nucleic acidsequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook and Russell, (2001), supra. Theso-called hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments or other oligonucleotides and may be labeledwith a detectable group such as 32P or any other detectable marker, suchas other radioisotopes, a fluorescent compound, an enzyme or an enzymeco-factor. Probes for hybridization can be made by labeling syntheticoligonucleotides based on the known PtIP-83 polypeptide-encoding nucleicacid sequence disclosed herein. Degenerate primers designed on the basisof conserved nucleotides or amino acid residues in the nucleic acidsequence or encoded amino acid sequence can additionally be used. Theprobe typically comprises a region of nucleic acid sequence thathybridizes under stringent conditions to at least about 12, at leastabout 25, at least about 50, 75, 100, 125, 150, 175 or 200 consecutivenucleotides of nucleic acid sequence encoding a PtIP-83 polypeptide ofthe disclosure or a fragment or variant thereof. Methods for thepreparation of probes for hybridization are generally known in the artand are disclosed in Sambrook and Russell, (2001), supra, hereinincorporated by reference.

For example, an entire nucleic acid sequence, encoding a PtIP-83polypeptide, disclosed herein or one or more portions thereof may beused as a probe capable of specifically hybridizing to correspondingnucleic acid sequences encoding PtIP-83 polypeptide-like sequences andmessenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique and arepreferably at least about 10 nucleotides in length or at least about 20nucleotides in length. Such probes may be used to amplify correspondingpesticidal sequences from a chosen organism by PCR. This technique maybe used to isolate additional coding sequences from a desired organismor as a diagnostic assay to determine the presence of coding sequencesin an organism. Hybridization techniques include hybridization screeningof plated DNA libraries (either plaques or colonies; see, for example,Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. “Stringent conditions” or “stringent hybridizationconditions” is used herein to refer to conditions under which a probewill hybridize to its target sequence to a detectably greater degreethan to other sequences (e.g., at least 2-fold over background).Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences that are 100%complementary to the probe can be identified (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, preferably less than 500 nucleotides inlength.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the Tm can be approximated from theequation of Meinkoth and Wahl, (1984) Anal. Biochem. 138:267-284:Tm=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is themolarity of monovalent cations, % GC is the percentage of guanosine andcytosine nucleotides in the DNA, % form is the percentage of formamidein the hybridization solution, and L is the length of the hybrid in basepairs. The Tm is the temperature (under defined ionic strength and pH)at which 50% of a complementary target sequence hybridizes to aperfectly matched probe. Tm is reduced by about 1° C. for each 1% ofmismatching; thus, Tm, hybridization, and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with 90% identity are sought, the Tm can be decreased 10°C. Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3or 4° C. lower than the thermal melting point (Tm); moderately stringentconditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10°C. lower than the thermal melting point (Tm); low stringency conditionscan utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or 20° C.lower than the thermal melting point (Tm). Using the equation,hybridization and wash compositions, and desired Tm, those of ordinaryskill will understand that variations in the stringency of hybridizationand/or wash solutions are inherently described. If the desired degree ofmismatching results in a Tm of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen, (1993)Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, N.Y.); and Ausubel, et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience,New York). See, Sambrook, et al., (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.).

In some embodiments polynucleotides are provided encoding a PtIP-83polypeptide comprising an amino acid sequence having at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% sequence identity to any one of SEQ ID NO:786-888.

In some embodiments polynucleotides are provided encoding a PtIP-83polypeptide comprising the amino acid sequence of any one of SEQ ID NO:786-888.

In some embodiments the polynucleotide encoding the PtIP-83 polypeptidecomprising the amino acid sequence of any one of SEQ ID NO: 786-888 is anon-genomic sequence.

In some embodiments the polynucleotide encoding the PtIP-83 polypeptidecomprising the amino acid sequence of any one of SEQ ID NO: 786-888 is acDNA.

Proteins and Variants and Fragments Thereof

PtIP-83 polypeptides are also encompassed by the disclosure.“Pteridophyta Insecticidal Protein-83” “PtIP-83 polypeptide”, and“PtIP-83 protein” as used herein interchangeably refers to a polypeptidehaving insecticidal activity including but not limited to insecticidalactivity against one or more insect pests of the Lepidoptera and/orColeoptera orders, and is sufficiently homologous to the protein of SEQID NO: 1. A variety of PtIP-83 polypeptides are contemplated. Sources ofPtIP-83 polypeptides or related proteins are fern species selected frombut not limited to Polypodium punctatum, Lygodium flexuosum, Microsorummusifolium, Adiantum peruvianum, Adiantum trapeziforme and Adiantumpedatum.

“Sufficiently homologous” is used herein to refer to an amino acidsequence that has at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence homology compared to a reference sequenceusing one of the alignment programs described herein using standardparameters. In some embodiments the sequence homology is against thefull length sequence of a PtIP-83 polypeptide. In some embodiments thePtIP-83 polypeptide has at least about 40%, 45%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater sequence identity compared to SEQ IDNO: 1. One of skill in the art will recognize that these values can beappropriately adjusted to determine corresponding homology of proteinstaking into account amino acid similarity and the like. In someembodiments the sequence identity is calculated using ClustalW algorithmin the ALIGNX® module of the Vector NTI® Program Suite (InvitrogenCorporation, Carlsbad, Calif.) with all default parameters. In someembodiments the sequence identity is across the entire length ofpolypeptide calculated using ClustalW algorithm in the ALIGNX® module ofthe Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, Calif.)with all default parameters.

As used herein, the terms “protein,” “peptide molecule,” or“polypeptide” includes any molecule that comprises five or more aminoacids. It is well known in the art that protein, peptide or polypeptidemolecules may undergo modification, including post-translationalmodifications, such as, but not limited to, disulfide bond formation,glycosylation, phosphorylation or oligomerization. Thus, as used herein,the terms “protein,” “peptide molecule” or “polypeptide” includes anyprotein that is modified by any biological or non-biological process.The terms “amino acid” and “amino acids” refer to all naturallyoccurring L-amino acids.

A “recombinant protein” or “recombinant polypeptide” is used herein torefer to a protein that is no longer in its natural environment, forexample in vitro or in a recombinant bacterial or plant host cell. A“purified protein” or “purified polypeptide” is used herein to refer toa protein that is substantially free of cellular material. A PtIP-83polypeptide that is substantially free of cellular material includespreparations of protein having less than about 30%, 20%, 10% or 5% (bydry weight) of non-pesticidal protein (also referred to herein as a“contaminating protein”).

“Fragments” or “biologically active portions” include polypeptidefragments comprising amino acid sequences sufficiently identical to aPtIP-83 polypeptide and that exhibit insecticidal activity. “Fragments”or “biologically active portions” of PtIP-83 polypeptides includesfragments comprising amino acid sequences sufficiently identical to theamino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ IDNO: 716, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757,SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ IDNO: 762, SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766,SEQ ID NO: 767, SEQ ID NO: 768 or SEQ ID NO: 769, wherein the PtIP-83polypeptide has insecticidal activity. Such biologically active portionscan be prepared by recombinant techniques and evaluated for insecticidalactivity. In some embodiments, the PtIP-83 polypeptide fragment is anN-terminal and/or a C-terminal truncation of at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27,28, 29, 30, 31, 32, 33, 34 or more amino acids from the N-terminusand/or C-terminus relative to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:716, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO:762, SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQID NO: 767, SEQ ID NO: 768 or SEQ ID NO: 769, e.g., by proteolysis, byinsertion of a start codon, by deletion of the codons encoding thedeleted amino acids and concomitant insertion of a start codon, and/orinsertion of a stop codon.

In some embodiments, the PtP-83 polypeptide fragments encompassed hereinresult from the removal of the N-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 or more amino acids relative to SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ IDNO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO:757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQID NO: 762, SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO:766, SEQ ID NO: 767, SEQ ID NO: 768, SEQ ID NO: 769 or variants thereof,e.g., by proteolysis or by insertion of a start codon, by deletion ofthe codons encoding the deleted amino acids and concomitant insertion ofa start codon.

“Variants” as used herein refers to proteins or polypeptides having anamino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the parental amino acidsequence.

In some embodiments a PtIP-83 polypeptide comprises an amino acidsequence having at least 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO:23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO:761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768 or SEQ ID NO: 769, whereinthe PtIP-83 polypeptide has insecticidal activity.

In some embodiments a PtIP-83 polypeptide comprises an amino acidsequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identityacross the entire length of the amino acid sequence of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,SEQ ID NO: 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755, SEQ IDNO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760,SEQ ID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID NO: 764, SEQ IDNO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768 or SEQ ID NO:769.

In some embodiments a PtIP-83 polypeptide comprises an amino acidsequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identityacross the entire length of the amino acid sequence of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,SEQ ID NO: 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755, SEQ IDNO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760,SEQ ID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID NO: 764, SEQ IDNO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768 or SEQ ID NO:769, and has at least one amino acid substitution, deletion, insertion,and/or addition at the N-terminus or C-terminus compared to the nativesequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ ID NO:754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ ID NO:763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQID NO: 768 or SEQ ID NO: 769.

In some embodiments a PtIP-83 polypeptide comprises an amino acidsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ ID NO:754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ ID NO:763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQID NO: 768 or SEQ ID NO: 769 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70 or more amino acid substitutions compared to thenative amino acid at the corresponding position of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ IDNO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQID NO: 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO:756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID NO:765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768 or SEQ ID NO: 769.

In some embodiments the sequence identity is across the entire length ofthe polypeptide calculated using ClustalW algorithm in the ALIGNX®module of the Vector NTI® Program Suite (Invitrogen Corporation,Carlsbad, Calif.) with all default parameters.

In some embodiments a PtIP-83 polypeptide comprises an amino acidsequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59 or 60 amino acid substitutions, in anycombination, compared to the native amino acid at the correspondingposition of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ ID NO:754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ ID NO:763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQID NO: 768 or SEQ ID NO: 769.

In some embodiments a PtIP-83 polypeptide comprises an amino acidsequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 amino acidsubstitutions, in any combination, compared to the native amino acid atthe corresponding position of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:716, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO:762, SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQID NO: 767, SEQ ID NO: 768 or SEQ ID NO: 769.

In some embodiments the PtIP-83 polypeptide comprises an amino acidsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ ID NO:754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ ID NO:763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQID NO: 768 or SEQ ID NO: 769.

In some embodiments the PtIP-83 polypeptide comprises a non-naturallyoccurring amino acid sequence. As used herein the term “non-naturallyoccurring amino acid sequence” means an amino acid sequence not found innature.

In some embodiments the PtIP-83 polypeptide is not the polypeptide ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ IDNO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759,SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ IDNO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768or SEQ ID NO: 769.

In some embodiments the PtIP-83 polypeptide is a variant of thepolypeptide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ IDNO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758,SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ IDNO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767,SEQ ID NO: 768 or SEQ ID NO: 769, wherein the PtIP-83 polypeptidevariant has at least one amino acid substitution, deletion, insertion,and/or addition at the N-terminus or C-terminus compared to SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ IDNO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755,SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ IDNO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID NO: 764,SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768 or SEQ IDNO: 769.

In some embodiments PtIP-83 polypeptide comprising an amino acidsequence of any one of SEQ ID NO: 236-299, SEQ ID NO: 334-367, SEQ IDNO: 398-427, SEQ ID NO: 518-607, SEQ ID NO: 640-645, and SEQ ID NO:728-737.

In some embodiments the PtIP-83 polypeptide is a variant of SEQ ID NO:1, wherein the amino acid at position 53 is Val, Ala, Cys or Thr; theamino acid at position 54 is Lys, Ala, Cys, Asp, Glu, Gly, His, Ile,Leu, Met, Asn, Gln, Arg, Ser or Thr; the amino acid at position 55 isArg, Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Gln, Ser, Thr,Val, Trp or Tyr; the amino acid at position 56 is Leu, Glu, Phe, Ile,Met, Thr or Val; the amino acid at position 57 is Tyr, Cys, Ile, Leu,Met, Thr or Val; the amino acid at position 58 is Val, Cys, Ile or Leu;the amino acid at position 59 is Phe, Leu, Met, Val or Tyr; the aminoacid at position 60 is Ala, Cys, Gly, Ser, Thr or Val; the amino acid atposition 61 is Asp, Glu, His or Ser; the amino acid at position 62 isVal, Ala, Cys, Ile, Leu or Thr; the amino acid at position 63 is Val,Ala, Cys, Ile, Leu, Met or Thr; the amino acid at position 64 is Glu,Ala, Cys, Phe, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val,Trp or Tyr; the amino acid at position 65 is Leu, Ala, Cys, Phe, His,Ile, Met, Asn, Gln, Thr, Val or Trp; the amino acid at position 66 isPro, Asp, Gly, Met, Gln or Arg; the amino acid at position 363 is Gln,Ala, Cys, Glu, Phe, Gly, His, Lys, Leu, Asn, Arg, Ser, Thr, Val or Trp;the amino acid at position 364 is Ile, Ala, Cys, Glu, Phe, His, Lys,Leu, Met, Asn, Gin, Ser, Thr, Val, Trp or Tyr; the amino acid atposition 365 is Leu, Ala, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Arg,Val, Trp or Tyr; the amino acid at position 366 is Gly, Ala, Cys, Phe,His, Ile, Lys, Leu, Met, Asn, Ser, Thr or Val; the amino acid atposition 367 is Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met,Asn, Pro, Gln, Arg, Thr, Val or Trp; the amino acid at position 368 isTyr, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro,Gln, Arg, Ser, Thr, Val or Trp; the amino acid at position 369 is Leu,Ala, Cys, Asp, Phe, Gly, Ile, Met, Thr or Val; the amino acid atposition 370 is Leu, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Met,Gln, Arg, Ser, Thr, Val, Trp or Tyr; the amino acid at position 371 isGln, Ala, Cys, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Asn, Arg, Ser, Thr,Val or Trp; the amino acid at position 372 is Gln, Ala, Cys, Asp, Phe,Gly, His, Ile, Leu, Asn, Arg, Ser, Val or Tyr; the amino acid atposition 373 is Asn, Ala, Cys, Asp, Phe, Gly, His, Ile, Lys, Gln, Ser,Thr, Val or Trp; the amino acid at position 556 is Trp, Phe, Thr or Tyr;the amino acid at position 557 is Arg, Cys, Asp, Gly, His, Ile, Lys,Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr; the amino acid atposition 558 is Ala, Cys, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro,Gln, Arg, Ser, Val, Trp or Tyr; the amino acid at position 559 is Lys,Ala, Cys, Phe, Gly, His, Ile, Leu, Asn, Gln, Arg, Ser, Thr, Val or Tyr;the amino acid at position 560 is Cys, Ala, Phe, Gly, Ile, Met, Asn,Arg, Ser, Thr or Val; the amino acid at position 561 is Lys, Ala, Cys,Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Arg, Ser, Thr, Val or Tyr;the amino acid at position 562 is Asn, Cys, Asp, Glu, Gly, His, Leu,Met, Arg, Ser, Thr, Val or Tyr; the amino acid at position 563 is Val,Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Asn, Gln, Thr or Trp; the aminoacid at position 564 is Ala, Cys, Gly, Met, Gln, Ser, Thr, Val, Trp orTyr; the amino acid at position 646 is Leu, Ala, Cys, Gly, Ile, Met,Asn, Gln, Ser, Thr or Val; the amino acid at position 647 is Leu, Asp,Gly, Met, Asn, Gln or Thr; the amino acid at position 648 is Met, Ala,Cys, Asp, Glu, Phe, Gly, His, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr,Val, Trp or Tyr; the amino acid at position 649 is Pro, Ala, Cys, Asp,Glu, Phe, Gly, His, Lys, Met, Asn, Gin, Arg, Ser, Thr, Trp or Tyr; theamino acid at position 650 is Thr, Ala, Cys, Asp, Phe, Gly, His, Ile,Lys, Leu, Met, Pro, Gln, Arg, Ser, Val or Tyr; the amino acid atposition 651 is Glu, Ala, Cys, Asp, Gly, His, Ile, Leu, Met, Asn, Pro,Gln, Arg, Ser, Thr, Val or Tyr; the amino acid at position 652 is Leu,Cys, Phe, Ile, Lys, Met, Pro, Arg, Ser, Thr or Val; the amino acid atposition 653 is Thr, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Pro,Arg, Ser, Val or Trp; the amino acid at position 654 is Thr, Ala, Cys,Phe, Ile, Lys, Leu, Met, Pro, Arg, Ser, Val, Trp or Tyr; the amino acidat position 655 is Trp, Phe or Tyr; the amino acid at position 771 isArg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Ser, Thr, Val,Trp or Tyr; the amino acid at position 772 is Arg, Ala, Cys, Asp, Glu,Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Ser, Thr, Val, Trp or Tyr;the amino acid at position 773 is Asp, Ala, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr; the amino acidat position 774 is Gln, Ala, Asp, Gly, His, Ile, Lys, Leu, Met, Asn,Pro, Arg, Ser, Thr, Val, Trp or Tyr; the amino acid at position 775 isVal, Ala, Cys, Asp, Glu, Gly, His, Ile, Asn, Pro, Gln, Arg, Ser, Thr orTyr; the amino acid at position 776 is Leu, Ala, Cys, Asp, Glu, Phe,Gly, His, Ile, Lys, Asn, Pro, Gln, Arg, Ser, Thr, Val or Tyr; the aminoacid at position 777 is Pro, Ala, Cys, Asp, Glu, Phe, Gly, His, Lys,Leu, Met, Asn, Gln, Ser, Thr, Val, Trp or Tyr; the amino acid atposition 778 is Phe, Ala, His, Ile, Leu, Met, Asn, Gin, Ser, Val, Trp orTyr; the amino acid at position 779 is Gln, Ala, Cys, Asp, Glu, Gly,His, Lys, Leu, Asn, Pro, Arg, Ser, Thr or Val; the amino acid atposition 780 is Ala, Cys, Asn, Pro, Gin or Ser; the amino acid atposition 781 is Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Asn, Gln, Arg,Ser, Thr, Val, Trp or Tyr; the amino acid at position 782 is Ala, Cys,Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Pro, Gln, Arg, Ser, Thr, Val,Trp or Tyr; the amino acid at position 783 is Pro, Ala, Cys, Asp, Glu,Gly, His, Asn, Gln, Arg, Ser, Thr or Val; the amino acid at position 784is Leu, Ala, Glu, Phe, His, Ile, Lys, Met, Asn, Pro, Gln, Ser, Thr, Valor Trp; the amino acid at position 785 is Asn, Ala, Cys, Glu, Phe, Gly,His, Ile, Lys, Leu, Met, Gln, Arg, Ser, Thr, Val, Trp or Tyr; and theamino acid at position 786 is Tyr, Phe, Ile, Leu or Trp.

In some embodiments the PtIP-83 polypeptide is a variant of SEQ ID NO:1, wherein the amino acid at position 1 is Met or deleted; the aminoacid at position 2 is Ala or deleted; the amino acid at position 3 isLeu, Val or deleted; the amino acid at position 4 is Val, Met or Leu;the amino acid at position 7 is Gly or Ser; the amino acid at position 8is Lys or Thr; the amino acid at position 10 is Phe or Tyr; the aminoacid at position 11 is Glu or Arg; the amino acid at position 18 is Metor Ile; the amino acid at position 19 is Gly, Pro or Ala; the amino acidat position 20 is Val or deleted; the amino acid at position 21 is Leuor Val; the amino acid at position 23 is Arg or Gln; the amino acid atposition 37 is Val or Leu; the amino acid at position 38 is Arg or Asn;the amino acid at position 40 is Ala or Ser; the amino acid at position43 is Asn or Asp; the amino acid at position 45 is Gly or Ala; the aminoacid at position 46 is Gln or Glu; the amino acid at position 48 is Glu,Pro or Val; the amino acid at position 51 is Glu or Gly; the amino acidat position 52 is Lys, Arg or Thr; the amino acid at position 56 is Leuor Val; the amino acid at position 59 is Phe or Leu; the amino acid atposition 66 is Pro or Ala; the amino acid at position 67 is Val, Pro orThr; the amino acid at position 68 is Val, Arg, Phe or Gly; the aminoacid at position 69 is Glu, Ala or Lys; the amino acid at position 70 isTrp, Thr, His, Tyr or Arg; the amino acid at position 71 is Arg, Pro ordeleted; the amino acid at position 72 is Trp, Asp, Leu or deleted; theamino acid at position 73 is Pro, Gln, Asn, His or deleted; the aminoacid at position 74 is Pro, Met or Thr; the amino acid at position 75 isGln, His or Arg; the amino acid at position 76 is Ile, Met or Leu; theamino acid at position 84 is Ile or Val; the amino acid at position 91is Trp or Phe; the amino acid at position 93 is Thr or Ile; the aminoacid at position 94 is Asp or Gly; the amino acid at position 96 is Argor Ser; the amino acid at position 97 is Gin, Phe or Arg; the amino acidat position 98 is Ser or deleted; the amino acid at position 99 is Aspor Ala; the amino acid at position 100 is Thr or Ala; the amino acid atposition 101 is Glu, Thr or Trp the amino acid at position 103 is His,Arg, Glu or Gln; the amino acid at position 105 is Thr or Pro; the aminoacid at position 108 is Lys, Gln or Glu; the amino acid at position 109is Leu or Val; the amino acid at position 111 is Ala or Thr; the aminoacid at position 112 is Ile, Arg, Thr or deleted; the amino acid atposition 113 is Gln, Ala, Gly or deleted; the amino acid at position 114is Arg, Glu or Ile; the amino acid at position 115 is Glu or Gln; theamino acid at position 116 is Glu, Asn, Gln or Arg; the amino acid atposition 117 is Asn, Val, Tyr or Phe; the amino acid at position 118 isArg or Lys; the amino acid at position 119 is Trp or Ser; the amino acidat position 122 is Thr, Lys or Ala; the amino acid at position 124 isAla or Thr; the amino acid at position 126 is Gly or Asp; the amino acidat position 127 is Met or Ala; the amino acid at position 128 is Asn orLys; the amino acid at position 131 is Val, Ile or Thr; the amino acidat position 133 is Ile or Val; the amino acid at position 134 is His orTyr; the amino acid at position 135 is Ala or Gly; the amino acid atposition 137 is Glu or Lys; the amino acid at position 139 is Gln orGlu; the amino acid at position 140 is Val, Arg or Leu; the amino acidat position 141 is Gly or Ser; the amino acid at position 142 is Val orPro; the amino acid at position 144 is Thr, Leu, Phe or Tyr; the aminoacid at position 145 is Met, Pro or Asn; the amino acid at position 146is Ser, Gly or Asn; the amino acid at position 147 is Trp or Asn; theamino acid at position 148 is Ser, Ala or Pro; the amino acid atposition 149 is Ser or deleted; the amino acid at position 150 is Val,lie or Tyr; the amino acid at position 152 is Arg, Ala, Val or Gly; theamino acid at position 154 is Ser, Trp or Glu; the amino acid atposition 156 is Leu, Asp or Gln; the amino acid at position 158 is Seror Cys; the amino acid at position 159 is Val, Thr or Ile; the aminoacid at position 162 is Ser or Ala; the amino acid at position 163 isGly or deleted; the amino acid at position 164 is Phe or deleted; theamino acid at position 165 is Arg or Ala; the amino acid at position 166is Ala, Arg, Met or Phe; the amino acid at position 167 is Val or His;the amino acid at position 168 is Ser or Asn; the amino acid at position169 is Val, His or Thr; the amino acid at position 170 is Phe or Val;the amino acid at position 171 is Glu, Asn or Asp; the amino acid atposition 172 is Val, Ala, Arg or Glu; the amino acid at position 175 isSer, Arg or Trp; the amino acid at position 176 is Val or Ile; the aminoacid at position 177 is Arg or Ile; the amino acid at position 179 isThr, Ile, Val or Ser; the amino acid at position 180 is Leu, Phe or Thr;the amino acid at position 181 is Gly, Thr, Gln or Ser; the amino acidat position 182 is Ala, Leu, Phe or Ile; the amino acid at position 183is Thr or Gly; the amino acid at position 184 is Leu, Thr, Ser or Arg;the amino acid at position 185 is Arg, Gly, Asp or Ala; the amino acidat position 186 is Pro, Val or Gln; the amino acid at position 187 isAsp, Thr or Ser; the amino acid at position 188 is His, Gly or Ala; theamino acid at position 189 is Ala, Arg, Pro or deleted; the amino acidat position 190 is Leu, Asn or deleted; the amino acid at position 191is Tyr or deleted; the amino acid at position 192 is Ser, Ile, Val orAsn; the amino acid at position 193 is Thr or Asp; the amino acid atposition 194 is Thr or Ser; the amino acid at position 195 is Met orThr; the amino acid at position 196 is Gln, His, Leu or Ser; the aminoacid at position 197 is Ala, Gly or Leu; the amino acid at position 198is Thr, Glu or Ala; the amino acid at position 199 is Pro or Arg; theamino acid at position 200 is Asn, Ser, Thr or Gly; the amino acid atposition 201 is Ala, Leu, Glu or Trp; the amino acid at position 202 isSer, Asp, Phe or Leu; the amino acid at position 203 is His, Pro, Gly orSer; the amino acid at position 204 is Ile, Trp, His or Gly; the aminoacid at position 205 is Ser, Asn or Ile; the amino acid at position 206is Ala, Gly, Asp, Tyr or Arg; the amino acid at position 207 is Phe, Valor Leu; the amino acid at position 208 is Asn, Ser, Pro or Leu; theamino acid at position 210 is Arg, Asp, Glu or Tyr; the amino acid atposition 211 is Ile, Ser or Thr; the amino acid at position 212 is Val,Ala or Asp; the amino acid at position 214 is Pro or Arg; the amino acidat position 215 is Ser or Thr; the amino acid at position 217 is Tyr orPhe; the amino acid at position 218 is Arg or Ser; the amino acid atposition 219 is Val or Ala; the amino acid at position 220 is Cys, Leuor Ser; the amino acid at position 221 is Pro or His; the amino acid atposition 222 is Leu, Arg or Ser; the amino acid at position 224 is Asnor Ser; the amino acid at position 225 is Asp, Arg or Thr; the aminoacid at position 226 is Thr or Asn; the amino acid at position 227 isAsp, Leu or deleted; the amino acid at position 228 is Thr or deleted;the amino acid at position 229 is Tyr or deleted; the amino acid atposition 230 is Leu or deleted; the amino acid at position 231 is Gly ordeleted; the amino acid at position 232 is lie or deleted; the aminoacid at position 233 is Pro or deleted; the amino acid at position 234is Ala, Pro or deleted; the amino acid at position 235 is Asp, lie orVal; the amino acid at position 236 is Val, Ser or Glu; the amino acidat position 237 is Ala, Phe or Tyr; the amino acid at position 238 isAla or Thr; the amino acid at position 239 is Val, Ser or Gly; the aminoacid at position 240 is Leu or Ile; the amino acid at position 243 isAsp or Glu; the amino acid at position 249 is Asn or Ser; the amino acidat position 252 is Leu or Met; the amino acid at position 257 is Thr orSer; the amino acid at position 259 is His or Leu; the amino acid atposition 266 is Ala or Val; the amino acid at position 267 is Cys orGly; the amino acid at position 268 is His, Arg or Tyr; the amino acidat position 272 is Asp or Glu; the amino acid at position 273 is Val,Met, lie or Leu; the amino acid at position 274 is Val or Met; the aminoacid at position 278 is Gly or Ala; the amino acid at position 279 isGlu or Val; the amino acid at position 281 is Leu or Ala; the amino acidat position 282 is Asn, Leu or Ile; the amino acid at position 285 isAsn or Ser; the amino acid at position 286 is Lys, Asp or Glu; the aminoacid at position 287 is Leu or Val; the amino acid at position 290 isPro, Gln or Arg; the amino acid at position 291 is Leu or Val; the aminoacid at position 292 is Lys or Val; the amino acid at position 293 isGlu or Gln; the amino acid at position 294 is Ser, Asn or Lys; the aminoacid at position 295 is Thr or Ser; the amino acid at position 296 isGln or His; the amino acid at position 297 is Leu or Met; the amino acidat position 300 is Ser or Thr; the amino acid at position 301 is Glu orAla; the amino acid at position 302 is Ser, Pro or Ala; the amino acidat position 304 is Lys or Asn; the amino acid at position 313 is Val orIle; the amino acid at position 314 is His, Glu or Gln; the amino acidat position 315 is Ala, Cys or Ser; the amino acid at position 316 isAla or Val; the amino acid at position 317 is Met or Ile; the amino acidat position 319 is Met or Ile; the amino acid at position 320 is Val orGly; the amino acid at position 321 is Arg or Pro; the amino acid atposition 322 is lie or Phe; the amino acid at position 323 is Gly orVal; the amino acid at position 324 is Leu or Ser; the amino acid atposition 336 is Ser or Asn; the amino acid at position 339 is Asn, Lysor Arg; the amino acid at position 350 is Arg or Gln; the amino acid atposition 351 is Glu or Asp; the amino acid at position 353 is Lys orArg; the amino acid at position 354 is Gln or Arg; the amino acid atposition 355 is Phe or Leu; the amino acid at position 356 is Lys orArg; the amino acid at position 360 is Ile, Val or Ala; the amino acidat position 365 is Leu or Phe; the amino acid at position 371 is or Glu;the amino acid at position 372 is or Lys; the amino acid at position 374is Arg or Lys; the amino acid at position 376 is Phe or Leu; the aminoacid at position 378 is Glu or Asp; the amino acid at position 381 isLeu or Val; the amino acid at position 388 is Ala or Ser; the amino acidat position 395 is Arg or Lys; the amino acid at position 396 is Glu,Gln or Gly; the amino acid at position 399 is Asp or Asn; the amino acidat position 400 is Asn, Thr or Asp; the amino acid at position 401 isThr or Ala; the amino acid at position 402 is Phe, lie or Leu; the aminoacid at position 406 is Asp or Glu; the amino acid at position 408 isLeu or Met; the amino acid at position 410 is Gly or Leu; the amino acidat position 414 is Ala or Glu; the amino acid at position 416 is Ser,Asn or Asp; the amino acid at position 417 is Ser, Arg or Gly; the aminoacid at position 423 is Lys or Gln; the amino acid at position 431 isArg or Lys; the amino acid at position 432 is Gln or Glu; the amino acidat position 436 is Arg or Glu; the amino acid at position 440 is Asn orArg; the amino acid at position 442 is Leu or Val; the amino acid atposition 447 is Ser, Lys or Arg; the amino acid at position 448 is Alaor Ser; the amino acid at position 451 is Gln or Met; the amino acid atposition 453 is Gly or Ala; the amino acid at position 455 is Ala orVal; the amino acid at position 457 is Leu or Val; the amino acid atposition 467 is Val or Ala; the amino acid at position 471 is Gly orAla; the amino acid at position 475 is Ser or Asn; the amino acid atposition 483 is Gly or Ala; the amino acid at position 493 is Gln orGly; the amino acid at position 504 is Val or Ile; the amino acid atposition 506 is Asp or His; the amino acid at position 509 is Asp orAsn; the amino acid at position 510 is Ser or Ala; the amino acid atposition 512 is Glu or Asp; the amino acid at position 515 is Gly orSer; the amino acid at position 516 is Gln or His; the amino acid atposition 517 is lie or Leu; the amino acid at position 519 is Asp, Glyor Gln; the amino acid at position 522 is Val, Glu, Pro or Val; theamino acid at position 525 is Glu or Asp; the amino acid at position 526is Leu or Met; the amino acid at position 539 is Val or Ile; the aminoacid at position 555 is Val or Ala; the amino acid at position 557 isArg or Lys; the amino acid at position 563 is Val or Met; the amino acidat position 571 is Ser or Cys; the amino acid at position 575 is Val orGlu; the amino acid at position 577 is Met or Ile; the amino acid atposition 579 is Glu or Gln; the amino acid at position 583 is Asp orGlu; the amino acid at position 589 is Met or Leu; the amino acid atposition 590 is Met or Leu; the amino acid at position 593 is Met orIle; the amino acid at position 595 is Arg or Gln; the amino acid atposition 596 is Ser or Thr; the amino acid at position 597 is Gln orHis; the amino acid at position 607 is Ala or Val; the amino acid atposition 608 is Asp or Asn; the amino acid at position 612 is Tyr, Hisor Phe; the amino acid at position 617 is Thr or Ile; the amino acid atposition 618 is Gln or His; the amino acid at position 625 is Arg orSer; the amino acid at position 626 is Met or Ile; the amino acid atposition 628 is Leu or Ile; the amino acid at position 633 is lie orMet; the amino acid at position 634 is Leu or Met; the amino acid atposition 642 is Arg or Met; the amino acid at position 648 is Met orThr; the amino acid at position 651 is Glu or Gln; the amino acid atposition 654 is Thr, Val or Ala; the amino acid at position 658 is Glyor Arg; the amino acid at position 663 is Gly or Ala; the amino acid atposition 664 is Asp or Asn; the amino acid at position 668 is Ala orThr; the amino acid at position 669 is Gln or His; the amino acid atposition 671 is Asn or Ser the amino acid at position 675 is Ile, Val orSer; the amino acid at position 678 is Met, Ile, Ala or Thr; the aminoacid at position 682 is Pro or Gln; the amino acid at position 683 isSer or Pro; the amino acid at position 685 is Asp or Asn; the amino acidat position 694 is Asp or Gly; the amino acid at position 697 is Asn orSer; the amino acid at position 704 is Glu or Gly; the amino acid atposition 714 is Ala or Gly; the amino acid at position 721 is Ser orPhe; the amino acid at position 722 is Ser or Asn; the amino acid atposition 724 is Ser or Thr; the amino acid at position 734 is His orGln; the amino acid at position 736 is Val or Ala; the amino acid atposition 737 is Lys or Gln; the amino acid at position 739 is Ala orSer; the amino acid at position 740 is Ser or Met; the amino acid atposition 741 is Gly or Asn; the amino acid at position 742 is lie orGly; the amino acid at position 743 is Gly or deleted; the amino acid atposition 745 is Gly or Asp; the amino acid at position 751 is Thr, Seror Ala; the amino acid at position 753 is Gln or Arg; the amino acid atposition 754 is Thr or Ser; the amino acid at position 756 is Thr orIle; the amino acid at position 757 is Val or Ile; the amino acid atposition 766 is lie or Val; the amino acid at position 773 is Asp orGlu; the amino acid at position 774 is Gln or Glu; the amino acid atposition 776 is Leu or Met; the amino acid at position 777 is Pro orThr; the amino acid at position 782 is Ala, Asp or Val; the amino acidat position 786 is Tyr or Phe; the amino acid at position 787 is His orGin; the amino acid at position 788 is Tyr or Met; the amino acid atposition 789 is Ala or Arg; the amino acid at position 790 is Tyr orThr; the amino acid at position 791 is Arg or Ala; the amino acid atposition 792 is Leu or Ser; the amino acid at position 796 is Asp orGlu; the amino acid at position 797 is Ser, Thr or Ala the amino acid atposition 802 is Glu or Gln; the amino acid at position 806 is Gln, Asp,Glu or His; the amino acid at position 810 is Lys or Thr; the amino acidat position 819 is Arg or His; the amino acid at position 829 is Lys,Ser, Ala or Pro; the amino acid at position 832 is Ala, Lys or Glu; theamino acid at position 833 is Gly or Glu; the amino acid at position 842is Leu or Pro; the amino acid at position 847 is Gln or Glu; the aminoacid at position 848 is Ile or Val; the amino acid at position 849 isVal or Ala; the amino acid at position 855 is Thr or Met; the amino acidat position 860 is Ile or Val; and the amino acid at position 864 is Hisor Gln.

In some embodiments the PtIP-83 polypeptide is a variant of SEQ ID NO:1, wherein the amino acid at position 1 is Met or deleted; the aminoacid at position 2 is Ala or deleted; the amino acid at position 3 isLeu, Val, Ile or deleted; the amino acid at position 4 is Val, Met, Ileor Leu; the amino acid at position 7 is Gly, Thr or Ser; the amino acidat position 8 is Lys, Arg, Ser or Thr; the amino acid at position 10 isPhe, Trp or Tyr; the amino acid at position 11 is Glu, Asp, Lys or Arg;the amino acid at position 18 is Met, Val, Leu or Ile; the amino acid atposition 19 is Gly, Pro or Ala; the amino acid at position 20 is Val,Ile, Leu or deleted; the amino acid at position 21 is Leu, Ile or Val;the amino acid at position 23 is Arg, Lys, Asn or Gln; the amino acid atposition 37 is Val, Ile or Leu; the amino acid at position 38 is Arg,Lys, Gln or Asn; the amino acid at position 40 is Ala, Gly, Thr or Ser;the amino acid at position 43 is Asn, Gin, Glu or Asp; the amino acid atposition 45 is Gly or Ala; the amino acid at position 46 is Gin, Asp,Asn or Glu; the amino acid at position 48 is Glu, Asp, Pro, Ile, Leu orVal; the amino acid at position 51 is Glu, Asp, Ala or Gly; the aminoacid at position 52 is Lys, Arg, Ser or Thr; the amino acid at position56 is Leu, Ile or Val; the amino acid at position 59 is Phe, Ile, Val orLeu; the amino acid at position 66 is Pro, Gly or Ala; the amino acid atposition 67 is Val, Pro, Ile, Leu, Ser or Thr; the amino acid atposition 68 is Val, Arg, Phe, Ile, Leu, Lys or Gly; the amino acid atposition 69 is Glu, Ala, Asp, Gly, Arg or Lys; the amino acid atposition 70 is Trp, Thr, His, Tyr, Lys or Arg; the amino acid atposition 71 is Arg, Pro, Lys or deleted; the amino acid at position 72is Trp, Asp, Leu, Ile, Val, Glu or deleted; the amino acid at position73 is Pro, Gln, Asn, His or deleted; the amino acid at position 74 isPro, Met, Ser or Thr; the amino acid at position 75 is Gln, His, Asn,Lys or Arg; the amino acid at position 76 is Ile, Met, Val or Leu; theamino acid at position 84 is Ile, Leu or Val; the amino acid at position91 is Trp or Phe; the amino acid at position 93 is Thr, Ser, Leu, Val orIle; the amino acid at position 94 is Asp, Glu, Ala or Gly; the aminoacid at position 96 is Arg, Lys, Thr or Ser; the amino acid at position97 is Gln, Phe, Asn, Lys or Arg; the amino acid at position 98 is Ser,Thr or deleted; the amino acid at position 99 is Asp, Glu, Gly or Ala;the amino acid at position 100 is Thr, Ser, Gly or Ala; the amino acidat position 101 is Glu, Thr, Asp, Ser or Trp the amino acid at position103 is His, Arg, Lys, Glu or Gln; the amino acid at position 105 is Thr,Ser or Pro; the amino acid at position 108 is Lys, Arg, Asn, Asp, Gln orGlu; the amino acid at position 109 is Leu, Ile or Val; the amino acidat position 111 is Ala, Ser or Thr; the amino acid at position 112 isIle, Arg, Thr, Leu, Val, Lys, Ser or deleted; the amino acid at position113 is Gln, Ala, Gly, Asn or deleted; the amino acid at position 114 isArg, Glu, Lys, Asp or Ile; the amino acid at position 115 is Glu, Asp,Asn or Gln; the amino acid at position 116 is Glu, Asn, Gln, Asp, Lys orArg; the amino acid at position 117 is Asn, Val, Tyr, Ile, Leu, Gln, Trpor Phe; the amino acid at position 118 is Arg or Lys; the amino acid atposition 119 is Trp, Thr or Ser; the amino acid at position 122 is Thr,Lys, Ser, Arg or Ala; the amino acid at position 124 is Ala, Gly, Ser orThr; the amino acid at position 126 is Gly, Ala, Glu or Asp; the aminoacid at position 127 is Met, Gly or Ala; the amino acid at position 128is Asn, Gln, Arg or Lys; the amino acid at position 131 is Val, Ile,Leu, Ser or Thr; the amino acid at position 133 is Ile, Leu or Val; theamino acid at position 134 is His or Tyr; the amino acid at position 135is Ala or Gly; the amino acid at position 137 is Glu, Asp, Arg or Lys;the amino acid at position 139 is Gln, Asn, Asp or Glu; the amino acidat position 140 is Val, Arg, Ile, Lys or Leu; the amino acid at position141 is Gly, Ala, Thr or Ser; the amino acid at position 142 is Val, Ile,Leu or Pro; the amino acid at position 144 is Thr, Leu, Phe, Ile, Val orTyr; the amino acid at position 145 is Met, Pro, Gln or Asn; the aminoacid at position 146 is Ser, Gly, Thr, Ala, Gln or Asn; the amino acidat position 147 is Trp, Gln, Tyr or Asn; the amino acid at position 148is Ser, Ala, Thr, Gly or Pro; the amino acid at position 149 is Ser, Thror deleted; the amino acid at position 150 is Val, Ile, Leu or Tyr; theamino acid at position 152 is Arg, Ala, Val, Ile, Leu, Lys or Gly; theamino acid at position 154 is Ser, Trp, Thr, Asp or Glu; the amino acidat position 156 is Leu, Asp, Ile, Val, Asn, Glu or Gln; the amino acidat position 158 is Ser, Thr or Cys; the amino acid at position 159 isVal, Thr, Leu or Ile; the amino acid at position 162 is Ser, Thr, Gly orAla; the amino acid at position 163 is Gly, Ala or deleted; the aminoacid at position 164 is Phe or deleted; the amino acid at position 165is Arg, Lys, Gly or Ala; the amino acid at position 166 is Ala, Arg,Met, Lys or Phe; the amino acid at position 167 is Val, Ile, Leu or His;the amino acid at position 168 is Ser, Thr, Gln or Asn; the amino acidat position 169 is Val, His, Ile, Leu, Ser or Thr; the amino acid atposition 170 is Phe, Ile, Leu or Val; the amino acid at position 171 isGlu, Asn, Gln or Asp; the amino acid at position 172 is Val, Ala, Arg,Ile, Leu, Gly, Lys, Asp or Glu; the amino acid at position 175 is Ser,Arg, Thr, Lys or Trp; the amino acid at position 176 is Val, Leu or Ile;the amino acid at position 177 is Arg, Lys, Leu, Val or Ile; the aminoacid at position 179 is Thr, Ile, Val, Leu or Ser; the amino acid atposition 180 is Leu, Phe, Ile, Val, Ser or Thr; the amino acid atposition 181 is Gly, Thr, Gln, Asn or Ser; the amino acid at position182 is Ala, Leu, Phe, Val or Ile; the amino acid at position 183 is Thr,Ser, Ala or Gly; the amino acid at position 184 is Leu, Thr, Ser, Ile,Val, Lys or Arg; the amino acid at position 185 is Arg, Gly, Asp, Lys,Glu or Ala; the amino acid at position 186 is Pro, Val, Ile, Leu, Asn orGln; the amino acid at position 187 is Asp, Thr, Glu or Ser; the aminoacid at position 188 is His, Gly or Ala; the amino acid at position 189is Ala, Arg, Pro, Lys, Gly or deleted; the amino acid at position 190 isLeu, Asn, Ile, Val, Gin or deleted; the amino acid at position 191 isTyr or deleted; the amino acid at position 192 is Ser, Ile, Val, Leu,Thr or Asn; the amino acid at position 193 is Thr, Ser, Glu or Asp; theamino acid at position 194 is Thr or Ser; the amino acid at position 195is Met or Thr; the amino acid at position 196 is Gln, His, Leu, Asn,Ile, Val, Thr or Ser; the amino acid at position 197 is Ala, Gly, Ile,Val or Leu; the amino acid at position 198 is Thr, Glu, Ser, Asp, Gly orAla; the amino acid at position 199 is Pro, Lys or Arg; the amino acidat position 200 is Asn, Ser, Thr, Gin, Ala or Gly; the amino acid atposition 201 is Ala, Leu, Glu, Ile, Asp or Trp; the amino acid atposition 202 is Ser, Asp, Phe, Ile, Val, Thr, Glu or Leu; the amino acidat position 203 is His, Pro, Gly, Ala, Thr or Ser; the amino acid atposition 204 is Ile, Trp, His, Leu, Val, Ala or Gly; the amino acid atposition 205 is Ser, Asn, Leu, Val, Thr, Gln or Ile; the amino acid atposition 206 is Ala, Gly, Asp, Tyr, Glu, Lys or Arg; the amino acid atposition 207 is Phe, Val, lie or Leu; the amino acid at position 208 isAsn, Ser, Pro, Gln, Thr, Val, lie or Leu; the amino acid at position 210is Arg, Asp, Glu, Lys, Ser or Tyr; the amino acid at position 211 isIle, Ser, Leu, Val or Thr; the amino acid at position 212 is Val, Ala,Ile, Leu, Glu, Gly or Asp; the amino acid at position 214 is Pro, Lys orArg; the amino acid at position 215 is Ser or Thr; the amino acid atposition 217 is Tyr or Phe; the amino acid at position 218 is Arg, Lys,Thr or Ser; the amino acid at position 219 is Val, Ile, Leu or Ala; theamino acid at position 220 is Cys, Leu, Ile, Val, Thr or Ser; the aminoacid at position 221 is Pro or His; the amino acid at position 222 isLeu, Arg, Lys, Ile, Val, Thr or Ser; the amino acid at position 224 isAsn, Gln, Thr or Ser; the amino acid at position 225 is Asp, Arg, Glu,Lys, Ser or Thr; the amino acid at position 226 is Thr, Ser, Gin or Asn;the amino acid at position 227 is Asp, Leu, Glu, Ile, Val or deleted;the amino acid at position 228 is Thr, Ser or deleted; the amino acid atposition 229 is Tyr or deleted; the amino acid at position 230 is Leu,Ile, Val or deleted; the amino acid at position 231 is Gly, Ala ordeleted; the amino acid at position 232 is Ile, Leu, Val or deleted; theamino acid at position 233 is Pro or deleted; the amino acid at position234 is Ala, Pro, Gly or deleted; the amino acid at position 235 is Asp,Ile, Leu, Glu or Val; the amino acid at position 236 is Val, Ser, Ile,Leu, Thr, Asp or Glu; the amino acid at position 237 is Ala, Phe or Tyr;the amino acid at position 238 is Ala, Gly, Ser or Thr; the amino acidat position 239 is Val, Ser, Ile, Leu, Thr, Ala or Gly; the amino acidat position 240 is Leu, Val or Ile; the amino acid at position 243 isAsp or Glu; the amino acid at position 249 is Asn, Gln, Thr or Ser; theamino acid at position 252 is Leu, lie, Val or Met; the amino acid atposition 257 is Thr or Ser; the amino acid at position 259 is His, Ile,Val or Leu; the amino acid at position 266 is Ala, Ile, Leu or Val; theamino acid at position 267 is Cys, Ala or Gly; the amino acid atposition 268 is His, Arg, Lys or Tyr; the amino acid at position 272 isAsp or Glu; the amino acid at position 273 is Val, Met, lie or Leu; theamino acid at position 274 is Val, Ile, Leu or Met; the amino acid atposition 278 is Gly or Ala; the amino acid at position 279 is Glu, Asp,Gly or Val; the amino acid at position 281 is Leu, lie, Val, Gly or Ala;the amino acid at position 282 is Asn, Leu or Ile; the amino acid atposition 285 is Asn, Gln, Thr or Ser; the amino acid at position 286 isLys, Asp, Arg or Glu; the amino acid at position 287 is Leu, lie or Val;the amino acid at position 290 is Pro, Gln, Asn, Lys or Arg; the aminoacid at position 291 is Leu, lie or Val; the amino acid at position 292is Lys, Arg, lie, Leu or Val; the amino acid at position 293 is Glu,Asp, Asn or Gln; the amino acid at position 294 is Ser, Asn, Thr, Gln,Arg or Lys; the amino acid at position 295 is Thr or Ser; the amino acidat position 296 is Gln, Asn or His; the amino acid at position 297 isLeu, Ile, Val or Met; the amino acid at position 300 is Ser or Thr; theamino acid at position 301 is Glu, Asp, Gly or Ala; the amino acid atposition 302 is Ser, Pro, Thr, Gly or Ala; the amino acid at position304 is Lys, Arg, Gln or Asn; the amino acid at position 313 is Val, Leuor Ile; the amino acid at position 314 is His, Glu, Asn, Asp or Gln; theamino acid at position 315 is Ala, Cys, Gly, Thr or Ser; the amino acidat position 316 is Ala, Ile, Leu or Val; the amino acid at position 317is Met, Leu, Val or Ile; the amino acid at position 319 is Met, Leu, Valor Ile; the amino acid at position 320 is Val, Ile, Leu, Ala or Gly; theamino acid at position 321 is Arg, Lys or Pro; the amino acid atposition 322 is Ile, Leu, Val or Phe; the amino acid at position 323 isGly, Ile, Leu or Val; the amino acid at position 324 is Leu, Ile, Val,Thr or Ser; the amino acid at position 336 is Ser, Thr, Gln or Asn; theamino acid at position 339 is Asn, Lys, Gln or Arg; the amino acid atposition 350 is Arg, Lys, Asn or Gln; the amino acid at position 351 isGlu or Asp; the amino acid at position 353 is Lys or Arg; the amino acidat position 354 is Gln, Asn, Lys or Arg; the amino acid at position 355is Phe, Ile, Leu or Leu; the amino acid at position 356 is Lys or Arg;the amino acid at position 360 is Ile, Val, Leu, Gly or Ala; the aminoacid at position 365 is Leu, lie, Val or Phe; the amino acid at position371 is or Glu or Asp; the amino acid at position 372 is or Lys or Arg;the amino acid at position 374 is Arg or Lys; the amino acid at position376 is Phe, Ile, Val or Leu; the amino acid at position 378 is Glu orAsp; the amino acid at position 381 is Leu, lie or Val; the amino acidat position 388 is Ala, Thr, Gly or Ser; the amino acid at position 395is Arg or Lys; the amino acid at position 396 is Glu, Gln, Asp, Asn, Alaor Gly; the amino acid at position 399 is Asp, Gln, Glu or Asn; theamino acid at position 400 is Asn, Thr, Ser, Glu, Gln or Asp; the aminoacid at position 401 is Thr, Ser, Gly or Ala; the amino acid at position402 is Phe, Ile, Val or Leu; the amino acid at position 406 is Asp orGlu; the amino acid at position 408 is Leu, Ile, Val or Met; the aminoacid at position 410 is Gly, Ile, Val, Ala or Leu; the amino acid atposition 414 is Ala, Gly, Asp or Glu; the amino acid at position 416 isSer, Asn, Thr, Gln, Glu or Asp; the amino acid at position 417 is Ser,Arg, Lys, Thr, Ala or Gly; the amino acid at position 423 is Lys, Arg,Asn or Gln; the amino acid at position 431 is Arg or Lys; the amino acidat position 432 is Gln, Asn, Asp or Glu; the amino acid at position 436is Arg, Lys, Asp or Glu; the amino acid at position 440 is Asn, Gln, Lysor Arg; the amino acid at position 442 is Leu, lie or Val; the aminoacid at position 447 is Ser, Lys, Thr or Arg; the amino acid at position448 is Ala, Gly, Thr or Ser; the amino acid at position 451 is Gln, Asnor Met; the amino acid at position 453 is Gly or Ala; the amino acid atposition 455 is Ala, Leu, lie or Val; the amino acid at position 457 isLeu, lie or Val; the amino acid at position 467 is Val, Ile, Leu, Gly orAla; the amino acid at position 471 is Gly or Ala; the amino acid atposition 475 is Ser, Thr, Gln or Asn; the amino acid at position 483 isGly or Ala; the amino acid at position 493 is Gln, Asn or Gly; the aminoacid at position 504 is Val, Leu or Ile; the amino acid at position 506is Asp, Glu or His; the amino acid at position 509 is Asp, Glu, Gln orAsn; the amino acid at position 510 is Ser, Thr, Gly or Ala; the aminoacid at position 512 is Glu or Asp; the amino acid at position 515 isGly, Ala, Thr or Ser; the amino acid at position 516 is Gln, Asn or His;the amino acid at position 517 is Ile, Val or Leu; the amino acid atposition 519 is Asp, Asn, Glu, Gly or Gln; the amino acid at position522 is Val, Glu, Pro, Ile, Leu or Asp; the amino acid at position 525 isGlu or Asp; the amino acid at position 526 is Leu, Ile, Val or Met; theamino acid at position 539 is Val, Leu or Ile; the amino acid atposition 555 is Val, Leu, lie or Ala; the amino acid at position 557 isArg or Lys; the amino acid at position 563 is Val, Leu, lie or Met; theamino acid at position 571 is Ser, Thr or Cys; the amino acid atposition 575 is Val, Leu, Ile, Asp or Glu; the amino acid at position577 is Met, Leu, Val or Ile; the amino acid at position 579 is Glu, Asp,Asn or Gln; the amino acid at position 583 is Asp or Glu; the amino acidat position 589 is Met, Ile, Val or Leu; the amino acid at position 590is Met, Ile, Val or Leu; the amino acid at position 593 is Met, Leu, Valor Ile; the amino acid at position 595 is Arg, Lys, Asn or Gln; theamino acid at position 596 is Ser or Thr; the amino acid at position 597is Gln, Asn or His; the amino acid at position 607 is Ala, Gly, Ile, Leuor Val; the amino acid at position 608 is Asp, Glu, Gln or Asn; theamino acid at position 612 is Tyr, His or Phe; the amino acid atposition 617 is Thr, Ser, Leu, Val or Ile; the amino acid at position618 is Gln, Asn or His; the amino acid at position 625 is Arg, Lys, Thror Ser; the amino acid at position 626 is Met, Leu, Val or Ile; theamino acid at position 628 is Leu, Val or Ile; the amino acid atposition 633 is Ile, Leu, Val or Met; the amino acid at position 634 isLeu, Ile, Val or Met; the amino acid at position 642 is Arg, Lys or Met;the amino acid at position 648 is Met, Ser or Thr; the amino acid atposition 651 is Glu, Asp, Asn or Gln; the amino acid at position 654 isThr, Val, Ser, Ile, Leu, Gly or Ala; the amino acid at position 658 isGly, Lys, Ala or Arg; the amino acid at position 663 is Gly or Ala; theamino acid at position 664 is Asp, Glu, Gln or Asn; the amino acid atposition 668 is Ala, Gly, Ser or Thr; the amino acid at position 669 isGln, Asn or His; the amino acid at position 671 is Asn, Gln, Thr or Serthe amino acid at position 675 is Ile, Val, Ile, Thr or Ser; the aminoacid at position 678 is Met, Ile, Ala, Leu, Ser or Thr; the amino acidat position 682 is Pro, Asn or Gln; the amino acid at position 683 isSer, Thr or Pro; the amino acid at position 685 is Asp, Glu, Asp or Asn;the amino acid at position 694 is Asp, Glu, Ala or Gly; the amino acidat position 697 is Asn, Gln, Thr or Ser; the amino acid at position 704is Glu, Asp, Ala or Gly; the amino acid at position 714 is Ala or Gly;the amino acid at position 721 is Ser, Thr or Phe; the amino acid atposition 722 is Ser, Thr, Gln or Asn; the amino acid at position 724 isSer or Thr; the amino acid at position 734 is His, Asn or Gln; the aminoacid at position 736 is Val, Leu, lie or Ala; the amino acid at position737 is Lys, Arg, Asn or Gln; the amino acid at position 739 is Ala, Gly,Thr or Ser; the amino acid at position 740 is Ser, Thr or Met; the aminoacid at position 741 is Gly, Ala, Gln or Asn; the amino acid at position742 is Ile, Leu, Val, Ala or Gly; the amino acid at position 743 is Glyor deleted; the amino acid at position 745 is Gly, Ala, Glu or Asp; theamino acid at position 751 is Thr, Ser, Gly or Ala; the amino acid atposition 753 is Gln, Asn, Lys or Arg; the amino acid at position 754 isThr or Ser; the amino acid at position 756 is Thr, Ser, Leu, Val or Ile;the amino acid at position 757 is Val, Leu or Ile; the amino acid atposition 766 is Ile, Leu or Val; the amino acid at position 773 is Aspor Glu; the amino acid at position 774 is Gln, Asn, Asp or Glu; theamino acid at position 776 is Leu, Ile, Val or Met; the amino acid atposition 777 is Pro, Ser or Thr; the amino acid at position 782 is Ala,Asp, Glu, Ile, Leu or Val; the amino acid at position 786 is Tyr or Phe;the amino acid at position 787 is His, Asn or Gln; the amino acid atposition 788 is Tyr or Met; the amino acid at position 789 is Ala, Lysor Arg; the amino acid at position 790 is Tyr or Thr; the amino acid atposition 791 is Arg, Lys, Gly or Ala; the amino acid at position 792 isLeu, Ile, Val, Thr or Ser; the amino acid at position 796 is Asp or Glu;the amino acid at position 797 is Ser, Thr or Ala the amino acid atposition 802 is Glu, Lys, Asp, Asn or Gln; the amino acid at position806 is Gin, Asp, Glu, Asn or His; the amino acid at position 810 is Lys,Arg or Thr; the amino acid at position 819 is Arg, Lys or His; the aminoacid at position 829 is Lys, Ser, Ala or Pro; the amino acid at position832 is Ala, Lys, Arg, Asp or Glu; the amino acid at position 833 is Gly,Ala, Asp or Glu; the amino acid at position 842 is Leu, Ile, Val or Pro;the amino acid at position 847 is Gin, Asn, Asp or Glu; the amino acidat position 848 is Ile, Leu or Val; the amino acid at position 849 isVal, Leu, Ile, Gly or Ala; the amino acid at position 855 is Thr, Ser orMet; the amino acid at position 860 is Ile, Leu or Val; the amino acidat position 864 is His, Asn or Gln;

In some embodiments the PtIP-83 polypeptide is a variant of SEQ ID NO:1, wherein the amino acid at position 1 is Met or deleted; the aminoacid at position 2 is Ala or deleted; the amino acid at position 3 isLeu, Val, Ile or deleted; the amino acid at position 4 is Val, Met, Ileor Leu; the amino acid at position 7 is Gly, Thr or Ser; the amino acidat position 8 is Lys, Arg, Ser or Thr; the amino acid at position 10 isPhe, Trp or Tyr; the amino acid at position 11 is Glu, Asp, Lys or Arg;the amino acid at position 18 is Met, Val, Leu or Ile; the amino acid atposition 19 is Gly, Pro or Ala; the amino acid at position 20 is Val,Ile, Leu or deleted; the amino acid at position 21 is Leu, Ile or Val;the amino acid at position 23 is Arg, Lys, Asn or Gln; the amino acid atposition 37 is Val, Ile or Leu; the amino acid at position 38 is Arg,Lys, Gln or Asn; the amino acid at position 40 is Ala, Gly, Thr or Ser;the amino acid at position 43 is Asn, Gin, Glu or Asp; the amino acid atposition 45 is Gly or Ala; the amino acid at position 46 is Gin, Asp,Asn or Glu; the amino acid at position 48 is Glu, Asp, Pro, Ile, Leu orVal; the amino acid at position 51 is Glu, Asp, Ala or Gly; the aminoacid at position 52 is Lys, Arg, Ser or Thr; the amino acid at position53 is Val, Ala, Cys or Thr; the amino acid at position 54 is Lys, Ala,Cys, Asp, Glu, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Ser or Thr; theamino acid at position 55 is Arg, Ala, Asp, Glu, Phe, Gly, His, Lys,Leu, Met, Asn, Gln, Ser, Thr, Val, Trp or Tyr; the amino acid atposition 56 is Leu, Glu, Phe, Ile, Met, Thr or Val; the amino acid atposition 57 is Tyr, Cys, Ile, Leu, Met, Thr or Val; the amino acid atposition 58 is Val, Cys, Ile or Leu; the amino acid at position 59 isPhe, Leu, Met, Val or Tyr; the amino acid at position 60 is Ala, Cys,Gly, Ser, Thr or Val; the amino acid at position 61 is Asp, Glu, His orSer; the amino acid at position 62 is Val, Ala, Cys, Ile, Leu or Thr;the amino acid at position 63 is Val, Ala, Cys, Ile, Leu, Met or Thr;the amino acid at position 64 is Glu, Ala, Cys, Phe, Gly, His, Ile, Leu,Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr; the amino acid atposition 65 is Leu, Ala, Cys, Phe, His, Ile, Met, Asn, Gln, Thr, Val orTrp; the amino acid at position 66 is Pro, Asp, Gly, Met, Gln or Arg;the amino acid at position 67 is Val, Pro, Ile, Leu, Ser or Thr; theamino acid at position 68 is Val, Arg, Phe, Ile, Leu, Lys or Gly; theamino acid at position 69 is Glu, Ala, Asp, Gly, Arg or Lys; the aminoacid at position 70 is Trp, Thr, His, Tyr, Lys or Arg; the amino acid atposition 71 is Arg, Pro, Lys or deleted; the amino acid at position 72is Trp, Asp, Leu, Ile, Val, Glu or deleted; the amino acid at position73 is Pro, Gln, Asn, His or deleted; the amino acid at position 74 isPro, Met, Ser or Thr; the amino acid at position 75 is Gln, His, Asn,Lys or Arg; the amino acid at position 76 is Ile, Met, Val or Leu; theamino acid at position 84 is Ile, Leu or Val; the amino acid at position91 is Trp or Phe; the amino acid at position 93 is Thr, Ser, Leu, Val orIle; the amino acid at position 94 is Asp, Glu, Ala or Gly; the aminoacid at position 96 is Arg, Lys, Thr or Ser; the amino acid at position97 is Gln, Phe, Asn, Lys or Arg; the amino acid at position 98 is Ser,Thr or deleted; the amino acid at position 99 is Asp, Glu, Gly or Ala;the amino acid at position 100 is Thr, Ser, Gly or Ala; the amino acidat position 101 is Glu, Thr, Asp, Ser or Trp the amino acid at position103 is His, Arg, Lys, Glu or Gln; the amino acid at position 105 is Thr,Ser or Pro; the amino acid at position 108 is Lys, Arg, Asn, Asp, Gln orGlu; the amino acid at position 109 is Leu, lie or Val; the amino acidat position 111 is Ala, Ser or Thr; the amino acid at position 112 isIle, Arg, Thr, Leu, Val, Lys, Ser or deleted; the amino acid at position113 is Gln, Ala, Gly, Asn or deleted; the amino acid at position 114 isArg, Glu, Lys, Asp or Ile; the amino acid at position 115 is Glu, Asp,Asn or Gln; the amino acid at position 116 is Glu, Asn, Gln, Asp, Lys orArg; the amino acid at position 117 is Asn, Val, Tyr, Ile, Leu, Gin, Trpor Phe; the amino acid at position 118 is Arg or Lys; the amino acid atposition 119 is Trp, Thr or Ser; the amino acid at position 122 is Thr,Lys, Ser, Arg or Ala; the amino acid at position 124 is Ala, Gly, Ser orThr; the amino acid at position 126 is Gly, Ala, Glu or Asp; the aminoacid at position 127 is Met, Gly or Ala; the amino acid at position 128is Asn, Gln, Arg or Lys; the amino acid at position 131 is Val, Ile,Leu, Ser or Thr; the amino acid at position 133 is Ile, Leu or Val; theamino acid at position 134 is His or Tyr; the amino acid at position 135is Ala or Gly; the amino acid at position 137 is Glu, Asp, Arg or Lys;the amino acid at position 139 is Gln, Asn, Asp or Glu; the amino acidat position 140 is Val, Arg, Ile, Lys or Leu; the amino acid at position141 is Gly, Ala, Thr or Ser; the amino acid at position 142 is Val, Ile,Leu or Pro; the amino acid at position 144 is Thr, Leu, Phe, Ile, Val orTyr; the amino acid at position 145 is Met, Pro, Gln or Asn; the aminoacid at position 146 is Ser, Gly, Thr, Ala, Gln or Asn; the amino acidat position 147 is Trp, Gln, Tyr or Asn; the amino acid at position 148is Ser, Ala, Thr, Gly or Pro; the amino acid at position 149 is Ser, Thror deleted; the amino acid at position 150 is Val, Ile, Leu or Tyr; theamino acid at position 152 is Arg, Ala, Val, Ile, Leu, Lys or Gly; theamino acid at position 154 is Ser, Trp, Thr, Asp or Glu; the amino acidat position 156 is Leu, Asp, Ile, Val, Asn, Glu or Gln; the amino acidat position 158 is Ser, Thr or Cys; the amino acid at position 159 isVal, Thr, Leu or Ile; the amino acid at position 162 is Ser, Thr, Gly orAla; the amino acid at position 163 is Gly, Ala or deleted; the aminoacid at position 164 is Phe or deleted; the amino acid at position 165is Arg, Lys, Gly or Ala; the amino acid at position 166 is Ala, Arg,Met, Lys or Phe; the amino acid at position 167 is Val, Ile, Leu or His;the amino acid at position 168 is Ser, Thr, Gln or Asn; the amino acidat position 169 is Val, His, Ile, Leu, Ser or Thr; the amino acid atposition 170 is Phe, Ile, Leu or Val; the amino acid at position 171 isGlu, Asn, Gln or Asp; the amino acid at position 172 is Val, Ala, Arg,Ile, Leu, Gly, Lys, Asp or Glu; the amino acid at position 175 is Ser,Arg, Thr, Lys or Trp; the amino acid at position 176 is Val, Leu or Ile;the amino acid at position 177 is Arg, Lys, Leu, Val or Ile; the aminoacid at position 179 is Thr, Ile, Val, Leu or Ser; the amino acid atposition 180 is Leu, Phe, Ile, Val, Ser or Thr; the amino acid atposition 181 is Gly, Thr, Gln, Asn or Ser; the amino acid at position182 is Ala, Leu, Phe, Val or Ile; the amino acid at position 183 is Thr,Ser, Ala or Gly; the amino acid at position 184 is Leu, Thr, Ser, Ile,Val, Lys or Arg; the amino acid at position 185 is Arg, Gly, Asp, Lys,Glu or Ala; the amino acid at position 186 is Pro, Val, Ile, Leu, Asn orGln; the amino acid at position 187 is Asp, Thr, Glu or Ser; the aminoacid at position 188 is His, Gly or Ala; the amino acid at position 189is Ala, Arg, Pro, Lys, Gly or deleted; the amino acid at position 190 isLeu, Asn, Ile, Val, Gln or deleted; the amino acid at position 191 isTyr or deleted; the amino acid at position 192 is Ser, Ile, Val, Leu,Thr or Asn; the amino acid at position 193 is Thr, Ser, Glu or Asp; theamino acid at position 194 is Thr or Ser; the amino acid at position 195is Met or Thr; the amino acid at position 196 is Gln, His, Leu, Asn,Ile, Val, Thr or Ser; the amino acid at position 197 is Ala, Gly, Ile,Val or Leu; the amino acid at position 198 is Thr, Glu, Ser, Asp, Gly orAla; the amino acid at position 199 is Pro, Lys or Arg; the amino acidat position 200 is Asn, Ser, Thr, Gln, Ala or Gly; the amino acid atposition 201 is Ala, Leu, Glu, lie, Asp or Trp; the amino acid atposition 202 is Ser, Asp, Phe, Ile, Val, Thr, Glu or Leu; the amino acidat position 203 is His, Pro, Gly, Ala, Thr or Ser; the amino acid atposition 204 is lie, Trp, His, Leu, Val, Ala or Gly; the amino acid atposition 205 is Ser, Asn, Leu, Val, Thr, Gln or Ile; the amino acid atposition 206 is Ala, Gly, Asp, Tyr, Glu, Lys or Arg; the amino acid atposition 207 is Phe, Val, lie or Leu; the amino acid at position 208 isAsn, Ser, Pro, Gln, Thr, Val, lie or Leu; the amino acid at position 210is Arg, Asp, Glu, Lys, Ser or Tyr; the amino acid at position 211 isIle, Ser, Leu, Val or Thr; the amino acid at position 212 is Val, Ala,Ile, Leu, Glu, Gly or Asp; the amino acid at position 214 is Pro, Lys orArg; the amino acid at position 215 is Ser or Thr; the amino acid atposition 217 is Tyr or Phe; the amino acid at position 218 is Arg, Lys,Thr or Ser; the amino acid at position 219 is Val, Ile, Leu or Ala; theamino acid at position 220 is Cys, Leu, Ile, Val, Thr or Ser; the aminoacid at position 221 is Pro or His; the amino acid at position 222 isLeu, Arg, Lys, Ile, Val, Thr or Ser; the amino acid at position 224 isAsn, Gln, Thr or Ser; the amino acid at position 225 is Asp, Arg, Glu,Lys, Ser or Thr; the amino acid at position 226 is Thr, Ser, Gln or Asn;the amino acid at position 227 is Asp, Leu, Glu, Ile, Val or deleted;the amino acid at position 228 is Thr, Ser or deleted; the amino acid atposition 229 is Tyr or deleted; the amino acid at position 230 is Leu,Ile, Val or deleted; the amino acid at position 231 is Gly, Ala ordeleted; the amino acid at position 232 is Ile, Leu, Val or deleted; theamino acid at position 233 is Pro or deleted; the amino acid at position234 is Ala, Pro, Gly or deleted; the amino acid at position 235 is Asp,Ile, Leu, Glu or Val; the amino acid at position 236 is Val, Ser, Ile,Leu, Thr, Asp or Glu; the amino acid at position 237 is Ala, Phe or Tyr;the amino acid at position 238 is Ala, Gly, Ser or Thr; the amino acidat position 239 is Val, Ser, Ile, Leu, Thr, Ala or Gly; the amino acidat position 240 is Leu, Val or Ile; the amino acid at position 243 isAsp or Glu; the amino acid at position 249 is Asn, Gln, Thr or Ser; theamino acid at position 252 is Leu, Ile, Val or Met; the amino acid atposition 257 is Thr or Ser; the amino acid at position 259 is His, Ile,Val or Leu; the amino acid at position 266 is Ala, lie, Leu or Val; theamino acid at position 267 is Cys, Ala or Gly; the amino acid atposition 268 is His, Arg, Lys or Tyr; the amino acid at position 272 isAsp or Glu; the amino acid at position 273 is Val, Met, lie or Leu; theamino acid at position 274 is Val, Ile, Leu or Met; the amino acid atposition 278 is Gly or Ala; the amino acid at position 279 is Glu, Asp,Gly or Val; the amino acid at position 281 is Leu, Ile, Val, Gly or Ala;the amino acid at position 282 is Asn, Leu or lie; the amino acid atposition 285 is Asn, Gln, Thr or Ser; the amino acid at position 286 isLys, Asp, Arg or Glu; the amino acid at position 287 is Leu, lie or Val;the amino acid at position 290 is Pro, Gln, Asn, Lys or Arg; the aminoacid at position 291 is Leu, lie or Val; the amino acid at position 292is Lys, Arg, Ile, Leu or Val; the amino acid at position 293 is Glu,Asp, Asn or Gln; the amino acid at position 294 is Ser, Asn, Thr, Gln,Arg or Lys; the amino acid at position 295 is Thr or Ser; the amino acidat position 296 is Gln, Asn or His; the amino acid at position 297 isLeu, Ile, Val or Met; the amino acid at position 300 is Ser or Thr; theamino acid at position 301 is Glu, Asp, Gly or Ala; the amino acid atposition 302 is Ser, Pro, Thr, Gly or Ala; the amino acid at position304 is Lys, Arg, Gln or Asn; the amino acid at position 313 is Val, Leuor Ile; the amino acid at position 314 is His, Glu, Asn, Asp or Gln; theamino acid at position 315 is Ala, Cys, Gly, Thr or Ser; the amino acidat position 316 is Ala, Ile, Leu or Val; the amino acid at position 317is Met, Leu, Val or Ile; the amino acid at position 319 is Met, Leu, Valor lie; the amino acid at position 320 is Val, Ile, Leu, Ala or Gly; theamino acid at position 321 is Arg, Lys or Pro; the amino acid atposition 322 is Ile, Leu, Val or Phe; the amino acid at position 323 isGly, Ile, Leu or Val; the amino acid at position 324 is Leu, Ile, Val,Thr or Ser; the amino acid at position 336 is Ser, Thr, Gln or Asn; theamino acid at position 339 is Asn, Lys, Gln or Arg; the amino acid atposition 350 is Arg, Lys, Asn or Gln; the amino acid at position 351 isGlu or Asp; the amino acid at position 353 is Lys or Arg; the amino acidat position 354 is Gln, Asn, Lys or Arg; the amino acid at position 355is Phe, Ile, Leu or Leu; the amino acid at position 356 is Lys or Arg;the amino acid at position 360 is Ile, Val, Leu, Gly or Ala; the aminoacid at position 363 is Gln, Ala, Cys, Glu, Phe, Gly, His, Lys, Leu,Asn, Arg, Ser, Thr, Val or Trp; the amino acid at position 364 is Ile,Ala, Cys, Glu, Phe, His, Lys, Leu, Met, Asn, Gln, Ser, Thr, Val, Trp orTyr; the amino acid at position 365 is Leu, Ala, Glu, Phe, Gly, His,Ile, Lys, Met, Asn, Arg, Val, Trp or Tyr; the amino acid at position 366is Gly, Ala, Cys, Phe, His, Ile, Lys, Leu, Met, Asn, Ser, Thr or Val;the amino acid at position 367 is Ser, Ala, Cys, Asp, Glu, Phe, Gly,His, Ile, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val or Trp; the amino acidat position 368 is Tyr, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys,Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp; the amino acid atposition 369 is Leu, Ala, Cys, Asp, Phe, Gly, Ile, Met, Thr or Val; theamino acid at position 370 is Leu, Ala, Cys, Asp, Glu, Phe, Gly, His,Ile, Lys, Met, Gln, Arg, Ser, Thr, Val, Trp or Tyr; the amino acid atposition 371 is Gln, Ala, Cys, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Asn,Arg, Ser, Thr, Val or Trp; the amino acid at position 372 is Gln, Ala,Cys, Asp, Phe, Gly, His, Ile, Leu, Asn, Arg, Ser, Val or Tyr; the aminoacid at position 373 is Asn, Ala, Cys, Asp, Phe, Gly, His, Ile, Lys,Gln, Ser, Thr, Val or Trp; the amino acid at position 374 is Arg or Lys;the amino acid at position 376 is Phe, Ile, Val or Leu; the amino acidat position 378 is Glu or Asp; the amino acid at position 381 is Leu,lie or Val; the amino acid at position 388 is Ala, Thr, Gly or Ser; theamino acid at position 395 is Arg or Lys; the amino acid at position 396is Glu, Gln, Asp, Asn, Ala or Gly; the amino acid at position 399 isAsp, Gln, Glu or Asn; the amino acid at position 400 is Asn, Thr, Ser,Glu, Gln or Asp; the amino acid at position 401 is Thr, Ser, Gly or Ala;the amino acid at position 402 is Phe, Ile, Val or Leu; the amino acidat position 406 is Asp or Glu; the amino acid at position 408 is Leu,Ile, Val or Met; the amino acid at position 410 is Gly, Ile, Val, Ala orLeu; the amino acid at position 414 is Ala, Gly, Asp or Glu; the aminoacid at position 416 is Ser, Asn, Thr, Gln, Glu or Asp; the amino acidat position 417 is Ser, Arg, Lys, Thr, Ala or Gly; the amino acid atposition 423 is Lys, Arg, Asn or Gln; the amino acid at position 431 isArg or Lys; the amino acid at position 432 is Gln, Asn, Asp or Glu; theamino acid at position 436 is Arg, Lys, Asp or Glu; the amino acid atposition 440 is Asn, Gin, Lys or Arg; the amino acid at position 442 isLeu, lie or Val; the amino acid at position 447 is Ser, Lys, Thr or Arg;the amino acid at position 448 is Ala, Gly, Thr or Ser; the amino acidat position 451 is Gln, Asn or Met; the amino acid at position 453 isGly or Ala; the amino acid at position 455 is Ala, Leu, lie or Val; theamino acid at position 457 is Leu, lie or Val; the amino acid atposition 467 is Val, Ile, Leu, Gly or Ala; the amino acid at position471 is Gly or Ala; the amino acid at position 475 is Ser, Thr, Gln orAsn; the amino acid at position 483 is Gly or Ala; the amino acid atposition 493 is Gln, Asn or Gly; the amino acid at position 504 is Val,Leu or lie; the amino acid at position 506 is Asp, Glu or His; the aminoacid at position 509 is Asp, Glu, Gln or Asn; the amino acid at position510 is Ser, Thr, Gly or Ala; the amino acid at position 512 is Glu orAsp; the amino acid at position 515 is Gly, Ala, Thr or Ser; the aminoacid at position 516 is Gln, Asn or His; the amino acid at position 517is Ile, Val or Leu; the amino acid at position 519 is Asp, Asn, Glu, Glyor Gln; the amino acid at position 522 is Val, Glu, Pro, Ile, Leu orAsp; the amino acid at position 525 is Glu or Asp; the amino acid atposition 526 is Leu, lie, Val or Met; the amino acid at position 539 isVal, Leu or Ile; the amino acid at position 555 is Val, Leu, lie or Ala;the amino acid at position 556 is Trp, Phe, Thr or Tyr; the amino acidat position 557 is Arg, Cys, Asp, Gly, His, Ile, Lys, Leu, Met, Asn,Pro, Gln, Ser, Thr, Val, Trp or Tyr; the amino acid at position 558 isAla, Cys, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser,Val, Trp or Tyr; the amino acid at position 559 is Lys, Ala, Cys, Phe,Gly, His, Ile, Leu, Asn, Gln, Arg, Ser, Thr, Val or Tyr; the amino acidat position 560 is Cys, Ala, Phe, Gly, Ile, Met, Asn, Arg, Ser, Thr orVal; the amino acid at position 561 is Lys, Ala, Cys, Asp, Glu, Phe,Gly, His, Ile, Leu, Met, Asn, Arg, Ser, Thr, Val or Tyr; the amino acidat position 562 is Asn, Cys, Asp, Glu, Gly, His, Leu, Met, Arg, Ser,Thr, Val or Tyr; the amino acid at position 563 is Val, Ala, Cys, Asp,Phe, His, Ile, Leu, Met, Asn, Gln, Thr or Trp; the amino acid atposition 564 is Ala, Cys, Gly, Met, Gln, Ser, Thr, Val, Trp or Tyr; theamino acid at position 571 is Ser, Thr or Cys; the amino acid atposition 575 is Val, Leu, Ile, Asp or Glu; the amino acid at position577 is Met, Leu, Val or Ile; the amino acid at position 579 is Glu, Asp,Asn or Gln; the amino acid at position 583 is Asp or Glu; the amino acidat position 589 is Met, Ile, Val or Leu; the amino acid at position 590is Met, Ile, Val or Leu; the amino acid at position 593 is Met, Leu, Valor Ile; the amino acid at position 595 is Arg, Lys, Asn or Gln; theamino acid at position 596 is Ser or Thr; the amino acid at position 597is Gln, Asn or His; the amino acid at position 607 is Ala, Gly, Ile, Leuor Val; the amino acid at position 608 is Asp, Glu, Gln or Asn; theamino acid at position 612 is Tyr, His or Phe; the amino acid atposition 617 is Thr, Ser, Leu, Val or lie; the amino acid at position618 is Gln, Asn or His; the amino acid at position 625 is Arg, Lys, Thror Ser; the amino acid at position 626 is Met, Leu, Val or Ile; theamino acid at position 628 is Leu, Val or Ile; the amino acid atposition 633 is Ile, Leu, Val or Met; the amino acid at position 634 isLeu, Ile, Val or Met; the amino acid at position 642 is Arg, Lys or Met;the amino acid at position 646 is Leu, Ala, Cys, Gly, Ile, Met, Asn,Gln, Ser, Thr or Val; the amino acid at position 647 is Leu, Asp, Gly,Met, Asn, Gln or Thr; the amino acid at position 648 is Met, Ala, Cys,Asp, Glu, Phe, Gly, His, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Val,Trp or Tyr; the amino acid at position 649 is Pro, Ala, Cys, Asp, Glu,Phe, Gly, His, Lys, Met, Asn, Gln, Arg, Ser, Thr, Trp or Tyr; the aminoacid at position 650 is Thr, Ala, Cys, Asp, Phe, Gly, His, Ile, Lys,Leu, Met, Pro, Gin, Arg, Ser, Val or Tyr; the amino acid at position 651is Glu, Ala, Cys, Asp, Gly, His, Ile, Leu, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val or Tyr; the amino acid at position 652 is Leu, Cys, Phe, lie,Lys, Met, Pro, Arg, Ser, Thr or Val; the amino acid at position 653 isThr, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Arg, Ser, Val orTrp; the amino acid at position 654 is Thr, Ala, Cys, Phe, Ile, Lys,Leu, Met, Pro, Arg, Ser, Val, Trp or Tyr; the amino acid at position 655is Trp, Phe or Tyr; the amino acid at position 658 is Gly, Lys, Ala orArg; the amino acid at position 663 is Gly or Ala; the amino acid atposition 664 is Asp, Glu, Gln or Asn; the amino acid at position 668 isAla, Gly, Ser or Thr; the amino acid at position 669 is Gln, Asn or His;the amino acid at position 671 is Asn, Gln, Thr or Ser the amino acid atposition 675 is Ile, Val, Ile, Thr or Ser; the amino acid at position678 is Met, Ile, Ala, Leu, Ser or Thr; the amino acid at position 682 isPro, Asn or Gln; the amino acid at position 683 is Ser, Thr or Pro; theamino acid at position 685 is Asp, Glu, Asp or Asn; the amino acid atposition 694 is Asp, Glu, Ala or Gly; the amino acid at position 697 isAsn, Gln, Thr or Ser; the amino acid at position 704 is Glu, Asp, Ala orGly; the amino acid at position 714 is Ala or Gly; the amino acid atposition 721 is Ser, Thr or Phe; the amino acid at position 722 is Ser,Thr, Gln or Asn; the amino acid at position 724 is Ser or Thr; the aminoacid at position 734 is His, Asn or Gln; the amino acid at position 736is Val, Leu, lie or Ala; the amino acid at position 737 is Lys, Arg, Asnor Gln; the amino acid at position 739 is Ala, Gly, Thr or Ser; theamino acid at position 740 is Ser, Thr or Met; the amino acid atposition 741 is Gly, Ala, Gln or Asn; the amino acid at position 742 isIle, Leu, Val, Ala or Gly; the amino acid at position 743 is Gly ordeleted; the amino acid at position 745 is Gly, Ala, Glu or Asp; theamino acid at position 751 is Thr, Ser, Gly or Ala; the amino acid atposition 753 is Gln, Asn, Lys or Arg; the amino acid at position 754 isThr or Ser; the amino acid at position 756 is Thr, Ser, Leu, Val or Ile;the amino acid at position 757 is Val, Leu or Ile; the amino acid atposition 766 is Ile, Leu or Val; the amino acid at position 771 is Arg,Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Ser, Thr, Val, Trp orTyr; the amino acid at position 772 is Arg, Ala, Cys, Asp, Glu, Phe,Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Ser, Thr, Val, Trp or Tyr; theamino acid at position 773 is Asp, Ala, Glu, Phe, Gly, His, Ile, Lys,Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr; the amino acid atposition 774 is Gln, Ala, Asp, Gly, His, Ile, Lys, Leu, Met, Asn, Pro,Arg, Ser, Thr, Val, Trp or Tyr; the amino acid at position 775 is Val,Ala, Cys, Asp, Glu, Gly, His, Ile, Asn, Pro, Gln, Arg, Ser, Thr or Tyr;the amino acid at position 776 is Leu, Ala, Cys, Asp, Glu, Phe, Gly,His, Ile, Lys, Asn, Pro, Gln, Arg, Ser, Thr, Val or Tyr; the amino acidat position 777 is Pro, Ala, Cys, Asp, Glu, Phe, Gly, His, Lys, Leu,Met, Asn, Gln, Ser, Thr, Val, Trp or Tyr; the amino acid at position 778is Phe, Ala, His, Ile, Leu, Met, Asn, Gin, Ser, Val, Trp or Tyr; theamino acid at position 779 is Gln, Ala, Cys, Asp, Glu, Gly, His, Lys,Leu, Asn, Pro, Arg, Ser, Thr or Val; the amino acid at position 780 isAla, Cys, Asn, Pro, Gin or Ser; the amino acid at position 781 is Ala,Cys, Asp, Glu, Phe, Gly, His, Ile, Asn, Gln, Arg, Ser, Thr, Val, Trp orTyr; the amino acid at position 782 is Ala, Cys, Asp, Glu, Phe, Gly,His, Ile, Lys, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr; the aminoacid at position 783 is Pro, Ala, Cys, Asp, Glu, Gly, His, Asn, Gln,Arg, Ser, Thr or Val; the amino acid at position 784 is Leu, Ala, Glu,Phe, His, Ile, Lys, Met, Asn, Pro, Gln, Ser, Thr, Val or Trp; the aminoacid at position 785 is Asn, Ala, Cys, Glu, Phe, Gly, His, Ile, Lys,Leu, Met, Gln, Arg, Ser, Thr, Val, Trp or Tyr; the amino acid atposition 786 is Tyr, Phe, Ile, Leu or Trp; the amino acid at position787 is His, Asn or Gln; the amino acid at position 788 is Tyr or Met;the amino acid at position 789 is Ala, Lys or Arg; the amino acid atposition 790 is Tyr or Thr; the amino acid at position 791 is Arg, Lys,Gly or Ala; the amino acid at position 792 is Leu, Ile, Val, Thr or Ser;the amino acid at position 796 is Asp or Glu; the amino acid at position797 is Ser, Thr or Ala the amino acid at position 802 is Glu, Lys, Asp,Asn or Gln; the amino acid at position 806 is Gln, Asp, Glu, Asn or His;the amino acid at position 810 is Lys, Arg or Thr; the amino acid atposition 819 is Arg, Lys or His; the amino acid at position 829 is Lys,Ser, Ala or Pro; the amino acid at position 832 is Ala, Lys, Arg, Asp orGlu; the amino acid at position 833 is Gly, Ala, Asp or Glu; the aminoacid at position 842 is Leu, Ile, Val or Pro; the amino acid at position847 is Gln, Asn, Asp or Glu; the amino acid at position 848 is Ile, Leuor Val; the amino acid at position 849 is Val, Leu, Ile, Gly or Ala; theamino acid at position 855 is Thr, Ser or Met; the amino acid atposition 860 is Ile, Leu or Val; and the amino acid at position 864 isHis, Asn or Gln.

In some embodiments the nucleic acid molecule encoding the PtIP-83polypeptide is derived from a fern species in the Division Pteridophyta.In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Class Psilotopsida. In some embodiments the PtIP-83polypeptide is derived from a fern species in the Class Psilotopsida,Order Psilotales. In some embodiments the PtIP-83 polypeptide is derivedfrom a fern species in the Class Psilotopsida, Order Ophioglossales. Insome embodiments the PtIP-83 polypeptide is derived from a fern speciesin the Class Psilotopsida, Order Ophioglossales, Family Psilotaceae. Insome embodiments the PtIP-83 polypeptide is derived from a fern speciesin the Class Psilotopsida, Order Ophioglossales Family Ophioglossaceae.In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Genus Ophioglossum L., Botrychium, Botrypus,Helminthostachys, Ophioderma, Cheiroglossa, Sceptridium or Mankyua. Insome embodiments the PtIP-83 polypeptide is derived from a species inthe Class Polypodiopsida/Pteridopsida. In some embodiments the PtIP-83polypeptide is derived from a fern species in the Order Osmundales(royal ferns); Family Osmundaceae. In some embodiments the PtIP-83polypeptide is derived from a fern species in the Order Hymenophyllales;Family Hymenophyllaceae. In some embodiments the PtIP-83 polypeptide isderived from a fern species in the Order Gleicheniales; FamilyGleicheniaceae, Family Dipteridaceae or Family Matoniaceae. In someembodiments the PtIP-83 polypeptide is derived from a fern species inthe Order Schizaeales; Family Lygodiaceae, Family Anemiaceae or FamilySchizaeaceae. In some embodiments the PtIP-83 polypeptide is derivedfrom a fern species in the Order Schizaeales; Family Schizaeaceae, GenusLygodium selected from but not limited to Lygodium articulatum, Lygodiumcircinatum, Lygodium conforme, Lygodium cubense, Lygodium digitatum,Lygodium flexuosum, Lygodium heterodoxum, Lygodium japonicum, Lygodiumkerstenii, Lygodium lanceolatum, Lygodium longifolium, Lygodiummerrilii, Lygodium micans, Lygodium microphyllum, Lygodiummicrostachyum, Lygodium oligostachyum, Lygodium palmatum, Lygodiumpolystachyum, Lygodium radiatum, Lygodium reticulatum, Lygodiumsalicifolium, Lygodium scandens, Lygodium smithianum, Lygodiumsubareolatum, Lygodium trifurcatum, Lygodium venustum, Lygodiumversteeghii, Lygodium volubile, and Lygodium yunnanense.

In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Order Salviniales; Family Marsileaceae or FamilySalviniaceae. In some embodiments the PtIP-83 polypeptide is derivedfrom a fern species in the Order Cyatheales; Family Thyrsopteridaceae,Family Loxsomataceae, Family Culcitaceae, Family Plagiogyriaceae, FamilyCibotiaceae, Family Cyatheaceae, Family Dicksoniaceae or FamilyMetaxyaceae.

In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Order Polypodiales; Family Lindsaeaceae, FamilySaccolomataceae, Family Cystodiaceae, Family Dennstaedtiaceae, FamilyPteridaceae, Family Aspleniaceae, Family Thelypteridaceae, FamilyWoodsiaceae, Family Onocleaceae, Family Blechnaceae, FamilyDryopteridaceae, Family Lomariopsidaceae, Family Tectariaceae, FamilyOleandraceae, Family Davalliaceae or Family Polypodiaceae.

In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Order Polypodiales, Family Pteridaceae, Genus Adiantaceaeselected from but not limited to Adiantum aethiopicum, Adiantumaleuticum, Adiantum bonatianum, Adiantum cajennense, Adiantumcapillus-junonis, Adiantum capillus-veneris, Adiantum caudatum, Adiantumchienii, Adiantum chilense, Adiantum cuneatum, Adiantum cunninghamii,Adiantum davidii, Adiantum diaphanum, Adiantum edentulum, Adiantumedgeworthii, Adiantum excisum, Adiantum fengianum, Adiantum fimbriatum,Adiantum flabellulatum, Adiantum formosanum, Adiantum formosum, Adiantumfulvum, Adiantum gravesii, Adiantum hispidulum, Adiantum induratum,Adiantum jordanii, Adiantum juxtapositum, Adiantum latifolium, Adiantumleveillei, Adiantum lianxianense, Adiantum malesianum, Adiantummariesii, Adiantum monochlamys, Adiantum myriosorum, Adiantum obliquum,Adiantum ogasawarense, Adiantum pedatum, Adiantum pentadactylon,Adiantum peruvianum, Adiantum philippense, Adiantum princeps, Adiantumpubescens, Adiantum raddianum, Adiantum reniforme, Adiantum roborowskii,Adiantum serratodentatum, Adiantum sinicum, Adiantum soboliferum,Adiantum subcordatum, Adiantum tenerum, Adiantum terminatum, Adiantumtetraphyllum, Adiantum trapeziforme, Adiantum venustum, Adiantumviridescens, and Adiantum viridimontanum.

In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Order Polypodiales, Family Aspleniaceae, Genus Asplenium.

In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Order Polypodiales, Family Aspleniaceae, Genus Aspleniumlselected from but not limited to Asplenium adiantum, Aspleniumadulterinum, Asplenium aequibasis, Asplenium aethiopicum, Aspleniumafricanum, Asplenium×alternifolium, Asplenium angustum, Aspleniumantiquum, Asplenium ascensionis, Asplenium attenuatum, Asplenium aureum,Asplenium auritum, Asplenium australasicum, Asplenium azoricum,Asplenium bifrons, Asplenium billottii, Asplenium bipinnatifidum,Asplenium brachycarpum, Asplenium bradleyi, Asplenium bulbiferum,Asplenium caudatum, Asplenium ceterach, Asplenium compressum, Aspleniumcongestum, Asplenium corderoanum, Asplenium crinicaule, Aspleniumcristatum, Asplenium cuneifolium, Asplenium cymbifolium, Aspleniumdaghestanicum, Asplenium dalhousiae, Asplenium dareoides, Aspleniumdaucifolium, Asplenium difforme, Asplenium fissum, Asplenium dimorphum,Asplenium divaricatum, Asplenium dregeanum, Asplenium×ebenoides,Asplenium ecuadorense, Asplenium feei Kunze, Asplenium fissum, Aspleniumflabellifolium, Asplenium flaccidum, Asplenium fontanum, Aspleniumforisiense, Asplenium formosum, Asplenium gemmiferum,Asplenium×germanicum, Asplenium gueinzii, Asplenium goudeyi, Aspleniumhemionitis, Asplenium hermannii-christii, Asplenium hookerianum,Asplenium hybridum, Asplenium incisum, Asplenium×jacksonii,Asplenium×kenzoi, Asplenium laciniatum, Asplenium lamprophyllum,Asplenium laserpitiifolium, Asplenium lepidum, Asplenium listeri,Asplenium longissimum, Asplenium lucidum, Asplenium lunulatum, Aspleniumlyallii, Asplenium macedonicum, Asplenium majoricum, Asplenium marinum,Asplenium×microdon, Asplenium milnei Carruth, Asplenium montanum,Asplenium musifolium, Asplenium nidus, Asplenium normale, Aspleniumobliquum, Asplenium oblongifolium, Asplenium obovatum, Aspleniumobtusatum, Asplenium oligolepidum, Asplenium oligophlebium, Aspleniumonopteris, Asplenium pacificum, Asplenium paleaceum, Asplenium palmeri,Asplenium petrarchae, Asplenium pinnatifidum, Asplenium planicaule,Asplenium platybasis, Asplenium platyneuron, Asplenium polyodon,Asplenium praemorsum, Asplenium prolongatum, Asplenium pteridoides,Asplenium resiliens, Asplenium rhizophyllum, Asplenium richardii,Asplenium ruprechtii, Asplenium ruta-muraria, Asplenium rustifolium,Asplenium sagittatum, Asplenium sandersonii, Asplenium×sarniense,Asplenium schizotrichum, Asplenium schweinfurthii, Aspleniumscleroprium, Asplenium scolopendrium (syn. Phyllitis scolopendrium),Asplenium seelosii, Asplenium septentrionale, Aspleniumseptentrionale×trichomanes, Asplenium serra, Asplenium serratum,Asplenium sessilifolium, Asplenium shuttleworthianum, Aspleniumsimplicifrons, Asplenium splendens, Asplenium surrogatum, Aspleniumtenerum, Asplenium terrestre, Asplenium theciferum, Aspleniumthunbergii, Asplenium trichomanes, Asplenium tutwilerae, Aspleniumvespertinum, Asplenium vieillardii, Asplenium virens, Asplenium viride,Asplenium vittiforme, and Asplenium viviparum.

In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Order Polypodiales, Family Blechnaceae, Genus Blecnum.

In some embodiments the the PtIP-83 polypeptide is derived from a fernspecies in the Order Polypodiales, Family Dryopteridaceae GenusAcrophorus, Genus Acrorumohra, Genus Anapausia, Genus Arachniodes, GenusBolbitis, Genus Ctenitis, Genus Cyclodium, Genus Cyrtogonellum, GenusCyrtomidictyum, Genus Cyrtomium, Genus Diacalpe, Genus Didymochlaena,Genus Dryopsis, Genus Dryopteris, Genus Elaphoglossum, GenusHypodematium, Genus Lastreopsis, Genus Leptorumohra, Genus Leucostegia,Genus Lithostegia, Genus Lomagramma, Genus Maxonia, Genus Megalastrum,Genus Olfersia, Genus Peranema, Genus Phanerophlebia, GenusPhanerophlebiopsis, Genus Polybotrya, Genus Polystichopsis, GenusPolystichum, Genus Rumohra, Genus Sorolepidium, Genus Stigmatopteris orGenus Teratophyllum.

In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Order Polypodiales, Family Dryopteridaceae, GenusPolystichum. In some embodiments the nucleic acid molecule encoding thePtIP-83 polypeptide is derived from a fern species in the OrderPolypodiales, Family Dryopteridaceae, Genus Polystichum selected frombut not limited to Polystichum acanthophyllum, Polystichumacrostichoides, Polystichum aculeatum, Polystichum acutidens,Polystichum acutipinnulum, Polystichum alcicorne, Polystichum aleuticum,Polystichum andersonii, Polystichum atkinsonii, Polystichumaustraliense, Polystichum bakerianum, Polystichum biaristatum,Polystichum bomiense, Polystichum bonseyi, Polystichum brachypterum,Polystichum braunii, Polystichum brachypterum, Polystichum calderonense,Polystichum californicum, Polystichum capillipes, Polystichum castaneum,Polystichum chilense, Polystichum christii Ching, Polystichum chuniiChing, Polystichum craspedosorum, Polystichum cyclolobum, Polystichumcystostegia, Polystichum deltodon, Polystichum dielsii, Polystichumdiscretum, Polystichum drepanum, Polystichum dudleyi, Polystichumduthiei, Polystichum echinatum, Polystichum erosum, Polystichumexcellens, Polystichum eximium, Polystichum falcatipinnum, Polystichumfalcinellum, Polystichum fallax, Polystichum formosanum, Polystichumgongboense, Polystichum grandifrons, Polystichum gymnocarpium,Polystichum haleakalense, Polystichum hancockii, Polystichumhecatopteron, Polystichum herbaceum, Polystichum imbricans, Polystichumincongruum, Polystichum kruckebergii, Polystichum kwakiutlii,Polystichum lachenense, Polystichum lanceolatum, Polystichum lemmonii,Polystichum lentum, Polystichum lonchitis, Polystichum longidens,Polystichum longipaleatum, Polystichum longipes, Polystichum luctuosum,Polystichum macleae, Polystichum macrochlaenum, Polystichum makinoi,Polystichum martini, Polystichum mayebarae, Polystichum mediocre,Polystichum medogense, Polystichum microchlamys, Polystichum mohrioides,Polystichum mollissimum, Polystichum monticola, Polystichum moorei,Polystichum morii, Polystichum moupinense, Polystichum muricatum,Polystichum nakenense, Polystichum neolobatum, Polystichum nepalense,Polystichum ningshenense, Polystichum obliquum, Polystichum omeiense,Polystichum ordinatum, Polystichum orientalitibeticum, Polystichumparamoupinense, Polystichum parvipinnulum, Polystichum piceopaleaceum,Polystichum polyblepharum, Polystichum prescottianum, Polystichumprionolepis, Polystichum proliferum, Polystichum pseudocastaneum,Polystichum pseudomakinoi, Polystichum punctiferum, Polystichum pungens,Polystichum qamdoense, Polystichum retrosopaleaceum, Polystichumrhombiforme, Polystichum rhomboidea, Polystichum richardii, Polystichumrigens, Polystichum rotundilobum, Polystichum scopulinum, Polystichumsemifertile, Polystichum setiferum, Polystichum setigerum, Polystichumshensiense, Polystichum silvaticum, Polystichum simplicipinnum,Polystichum sinense, Polystichum squarrosum, Polystichum stenophyllum,Polystichum stimulans, Polystichum submite, Polystichum tacticopterum,Polystichum thomsoni, Polystichum tibeticum, Polystichum transvaalense,Polystichum tripteron, Polystichum tsus-simense, Polystichum vestitum,Polystichum wattii, Polystichum whiteleggei, Polystichum xiphophyllum,Polystichum yadongense, and Polystichum yunnanense.

In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Order Polypodiales, Family Dryopteridaceae, GenusRumohra. In some embodiments the nucleic acid molecule encoding thePtIP-83 polypeptide is derived from a fern species in the OrderPolypodiales, Family Dryopteridaceae, Genus Rumohra selected from butnot limited to Rumohra adiantiformis, Rumohra aristata, Rumohrabartonae, Rumohra berteroana, Rumohra capuronii, Rumohra glandulosa,Rumohra humbertii, Rumohra linearisquamosa, Rumohra lokohensis, Rumohramadagascarica, and Rumohra quadrangularis.

In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Order Polypodiales, Family Lomariopsidaceae, GenusNephrolepis.

In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Order Polypodiales, Family Polypodiaceae, GenusCampyloneurum, Genus Drynaria, Genus Lepisorus, Genus Microgramma, GenusMicrosorum, Genus Neurodium, Genus Niphidium, Genus Pecluma M.G., GenusPhlebodium, Genus Phymatosorus, Genus Platycerium, Genus Pleopeltis,Genus Polypodium.

In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Order Polypodiales, Family Polypodiaceae, GenusMicrosorum.

In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Order Polypodiales, Family Polypodiaceae, GenusMicrosorum selected from but not limited to Microsorum alatum,Microsorum angustifolium, Microsorum aurantiacum, Microsorumaustraliense, Microsorum baithoense, Microsorum basicordatum, Microsorumbiseriatum, Microsorum brassii, Microsorum buergerianum, Microsorumchapaense, Microsorum cinctum, Microsorum commutatum, Microsorumcongregatifolium, Microsorum cuneatum, Microsorum cuspidatum, Microsorumdengii, Microsorum egregium, Microsorum emeiensis, Microsorum ensatum,Microsorum ensiforme, Microsorum excelsum, Microsorum fortunei,Microsorum griseorhizoma, Microsorum grossum, Microsorum hemionitideum,Microsorum henryi, Microsorum heterocarpum, Microsorum heterolobum,Microsorum howense, Microsorum insigne, Microsorum intermedium,Microsorum kongtingense, Microsorum krayanense, Microsorum lanceolatum,Microsorum lancifolium, Microsorum lastii, Microsorum latilobatum,Microsorum leandrianum, Microsorum lineare, Microsorum linguiforme,Microsorum longissimum, Microsorum longshengense, Microsorum maculosum,Microsorum maximum, Microsorum membranaceum, Microsorum membranifolium,Microsorum microsorioides, Microsorum minor, Microsorum monstrosum,Microsorum muliense, Microsorum mutense, Microsorum nanchuanense,Microsorum ningpoense, Microsorum normale, Microsorum novae-zealandiae,Microsorum ovalifolium, Microsorum ovatum, Microsorum palmatopedatum,Microsorum pappei, Microsorum papuanum, Microsorum parksii, Microsorumpentaphyllum, Microsorum piliferum, Microsorum pitcairnense, Microsorumpowellii, Microsorum pteropodum, Microsorum pteropus, Microsorumpunctatum, Microsorum pustulatum, Microsorum rampans, Microsorumrevolutum, Microsorum rubidum, Microsorum samarense, Microsorumsapaense, Microsorum sarawakense, Microsorum scandens, Microsorumscolopendria, Microsorum sibomense, Microsorum sinense, Microsorumsopuense, Microsorum spectrum, Microsorum steerei, Microsorumsubhemionitideum, Microsorum submarginale, Microsorum subnudum,Microsorum superficiale, Microsorum takhtajanii, Microsorum tenuipes,Microsorum tibeticum, Microsorum triglossum, Microsorum truncatum,Microsorum tsaii, Microsorum varians, Microsorum venosum, Microsorumvieillardii, Microsorum×inaequibasis, Microsorum yiliangensis, andMicrosorum zippelii.

In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Order Polypodiales, Family Polypodiaceae, GenusPolypodium L.

In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Order Polypodiales, Family Polypodiaceae, GenusPolypodium L. selected from but not limited to Polypodium absidatum,Polypodium acutifolium, Polypodium adiantiforme, Polypodium aequale,Polypodium affine, Polypodium albidopaleatum, Polypodium alcicorne,Polypodium alfarii, Polypodium alfredii, Polypodium alfredii var.curtii, Polypodium allosuroides, Polypodium alsophilicola, Polypodiumamamianum, Polypodium amoenum, Polypodium amorphum, Polypodiumanetioides, Polypodium anfractuosum, Polypodium anguinum, Polypodiumangustifolium f. remotifolia, Polypodium angustifolium var. amphostenon,Polypodium angustifolium var. heterolepis, Polypodium angustifolium var.monstrosa, Polypodium angustipaleatum, Polypodium angustissimum,Polypodium anisomeron var. pectinatum, Polypodium antioquianum,Polypodium aoristisorum, Polypodium apagolepis, Polypodium apicidens,Polypodium apiculatum, Polypodium apoense, Polypodium appalachianum,Polypodium appressum, Polypodium arenarium, Polypodium argentinum,Polypodium argutum, Polypodium armatum, Polypodium aromaticum,Polypodium aspersum, Polypodium assurgens, Polypodium atrum, Polypodiumauriculatum, Polypodium balaonense, Polypodium balliviani, Polypodiumbamleri, Polypodium bangii, Polypodium bartlettii, Polypodium basale,Polypodium bernoullii, Polypodium biauritum, Polypodium bifrons,Polypodium blepharodes, Polypodium bolivari, Polypodium bolivianum,Polypodium bolobense, Polypodium bombycinum, Polypodium bombycinum var.insularum, Polypodium bradeorum, Polypodium bryophilum, Polypodiumbryopodum, Polypodium buchtienii, Polypodium buesii, Polypodiumbulbotrichum, Polypodium caceresii, Polypodium californicum f.brauscombii, Polypodium californicum f. parsonsiae, Polypodiumcalifornicum, Polypodium calophlebium, Polypodium calvum, Polypodiumcamptophyllarium var. abbreviatum, Polypodium capitellatum, Polypodiumcarpinterae, Polypodium chachapoyense, Polypodium chartaceum, Polypodiumchimantense, Polypodium chiricanum, Polypodium choquetangense,Polypodium christensenii, Polypodium christii, Polypodium chrysotrichum,Polypodium ciliolepis, Polypodium cinerascens, Polypodium collinsii,Polypodium colysoides, Polypodium confluens, Polypodium conforme,Polypodium confusum, Polypodium congregatifolium, Polypodium connellii,Polypodium consimile var. bourgaeanum, Polypodium consimile var. minor,Polypodium conterminans, Polypodium contiguum, Polypodium cookii,Polypodium coriaceum, Polypodium coronans, Polypodium costaricense,Polypodium costatum, Polypodium crassifolium f. angustissimum,Polypodium crassifolium var. longipes, Polypodium crassulum, Polypodiumcraterisorum, Polypodium cryptum, Polypodium crystalloneuron, Polypodiumcucullatum var. planum, Polypodium cuencanum, Polypodium cumingianum,Polypodium cupreolepis, Polypodium curranii, Polypodium curvans,Polypodium cyathicola, Polypodium cyathisorum, Polypodium cyclocolpon,Polypodium daguense, Polypodium damunense, Polypodium dareiformioides,Polypodium dasypleura, Polypodium decipiens, Polypodium decorum,Polypodium delicatulum, Polypodium deltoideum, Polypodium demeraranum,Polypodium denticulatum, Polypodium diaphanum, Polypodium dilatatum,Polypodium dispersum, Polypodium dissectum, Polypodium dissimulans,Polypodium dolichosorum, Polypodium dolorense, Polypodiumdonnell-smithii, Polypodium drymoglossoides, Polypodium ebeninum,Polypodium eggersii, Polypodium elmeri, Polypodium elongatum, Polypodiumenterosoroides, Polypodium erubescens, Polypodium erythrolepis,Polypodium erythrotrichum, Polypodium eurybasis, Polypodium eurybasisvar. villosum, Polypodium exornans, Polypodium falcoideum, Polypodiumfallacissimum, Polypodium farinosum, Polypodium faucium, Polypodiumfeei, Polypodium ferrugineum, Polypodium feuillei, Polypodium firmulum,Polypodium firmum, Polypodium flaccidum, Polypodium flagellare,Polypodium flexuosum, Polypodium flexuosum var. ekmanii, Polypodiumforbesii, Polypodium formosanum, Polypodium fraxinifolium subsp.articulatum, Polypodium fraxinifolium subsp. luridum, Polypodiumfructuosum, Polypodium fucoides, Polypodium fulvescens, Polypodiumgaleottii, Polypodium glaucum, Polypodium glycyrrhiza, Polypodiumgracillimum, Polypodium gramineum, Polypodium grandifolium, Polypodiumgratum, Polypodium graveolens, Polypodium griseo-nigrum, Polypodiumgriseum, Polypodium guttatum, Polypodium haalilioanum, Polypodiumhammatisorum, Polypodium hancockii, Polypodium haplophlebicum,Polypodium harrisii, Polypodium hastatum var. simplex, Polypodiumhawaiiense, Polypodium heanophyllum, Polypodium helleri, Polypodiumhemionitidium, Polypodium henryi, Polypodium herzogii, Polypodiumhesperium, Polypodium hessii, Polypodium hombersleyi, Polypodiumhostmannii, Polypodium humile, Polypodium hyalinum, Polypodium iboense,Polypodium induens var. subdentatum, Polypodium insidiosum, Polypodiuminsigne, Polypodium intermedium subsp. masafueranum var. obtuseserratum,Polypodium intramarginale, Polypodium involutum, Polypodium itatiayense,Polypodium javanicum, Polypodium juglandifolium, Polypodium kaniense,Polypodium knowltoniorum, Polypodium kyimbilense, Polypodiuml'herminieri var. costaricense, Polypodium lachniferum f. incurvata,Polypodium lachniferum var. glabrescens, Polypodium lachnopus,Polypodium lanceolatum var. complanatum, Polypodium lanceolatum var.trichophorum, Polypodium latevagans, Polypodium laxifrons, Polypodiumlaxifrons var. lividum, Polypodium lehmannianum, Polypodium leiorhizum,Polypodium leptopodon, Polypodium leuconeuron var. angustifolia,Polypodium leuconeuron var. latifolium, Polypodium leucosticta,Polypodium limulum, Polypodium lindigii, Polypodium lineatum, Polypodiumlomarioides, Polypodium longifrons, Polypodium loretense, Polypodiumloriceum var. umbraticum, Polypodium loriforme, Polypodium loxogramme f.gigas, Polypodium ludens, Polypodium luzonicum, Polypodium lycopodioidesf. obtusum, Polypodium lycopodioides L., Polypodium macrolepis,Polypodium macrophyllum, Polypodium macrosorum, Polypodiummacrosphaerum, Polypodium maculosum, Polypodium madrense, Polypodiummanmeiense, Polypodium margaritiferum, Polypodium maritimum, Polypodiummartensii, Polypodium mayoris, Polypodium megalolepis, Polypodiummelanotrichum, Polypodium menisciifolium var. pubescens, Polypodiummeniscioides, Polypodium merrillii, Polypodium mettenii, Polypodiummexiae, Polypodium microsorum, Polypodium militare, Polypodium minimum,Polypodium minusculum, Polypodium mixtum, Polypodium mollendense,Polypodium mollissimum, Polypodium moniliforme var. minus, Polypodiummonoides, Polypodium monticola, Polypodium montigenum, Polypodiummoritzianum, Polypodium moultonii, Polypodium multicaudatum, Polypodiummultilineatum, Polypodium multisorum, Polypodium munchii, Polypodiummuscoides, Polypodium myriolepis, Polypodium myriophyllum, Polypodiummyriotrichum, Polypodium nematorhizon, Polypodium nemorale, Polypodiumnesioticum, Polypodium nigrescentium, Polypodium nigripes, Polypodiumnigrocinctum, Polypodium nimbatum, Polypodium nitidissimum, Polypodiumnitidissimum var. latior, Polypodium nubrigenum, Polypodium oligolepis,Polypodium oligosorum, Polypodium oligosorum, Polypodium olivaceum,Polypodium olivaceum var. elatum, Polypodium oodes, Polypodiumoosphaerum, Polypodium oreophilum, Polypodium ornatissimum, Polypodiumornatum, Polypodium ovatum, Polypodium oxylobum, Polypodium oxypholis,Polypodium pakkaense, Polypodium pallidum, Polypodium palmatopedatum,Polypodium palmeri, Polypodium panamense, Polypodium parvum, Polypodiumpatagonicum, Polypodium paucisorum, Polypodium pavonianum, Polypodiumpectinatum var. caliense, Polypodium pectinatum var. hispidum,Polypodium pellucidum, Polypodium pendulum var. boliviense, Polypodiumpercrassum, Polypodium perpusillum, Polypodium peruvianum var.subgibbosum, Polypodium phyllitidis var. elongatum, Polypodiumpichinchense, Polypodium pilosissimum, Polypodium pilosissimum var.glabriusculum, Polypodium pilossimum var. tunguraquensis, Polypodiumpityrolepis, Polypodium platyphyllum, Polypodium playfairii, Polypodiumplebeium var. cooperi, Polypodium plectolepidioides, Polypodiumpleolepis, Polypodium plesiosorum vari, Polypodium podobasis, Polypodiumpodocarpum, Polypodium poloense, Polypodium polydatylon, Polypodiumpolypodioides var. aciculare, Polypodium polypodioides var.michauxianum, Polypodium praetermissum, Polypodium preslianum var.immersum, Polypodium procerum, Polypodium procerum, Polypodiumproductum, Polypodium productum, Polypodium prolongilobum, Polypodiumpropinguum, Polypodium proteus, Polypodium pruinatum, Polypodiumpseudocapillare, Polypodium pseudofraternum, Polypodium pseudonutans,Polypodium pseudoserratum, Polypodium pulcherrimum, Polypodiumpulogense, Polypodium pungens, Polypodium purpusii, Polypodium radicale,Polypodium randallii, Polypodium ratiborii, Polypodium reclinatum,Polypodium recreense, Polypodium repens var. abruptum, Polypodiumrevolvens, Polypodium rhachipterygium, Polypodium rhomboideum,Polypodium rigens, Polypodium robustum, Polypodium roraimense,Polypodium roraimense, Polypodium rosei, Polypodium rosenstockii,Polypodium rubidum, Polypodium rudimentum, Polypodium rusbyi, Polypodiumsablanianum, Polypodium sarmentosum, Polypodium saxicola, Polypodiumschenckii, Polypodium schlechteri, Polypodium scolopendria, Polypodiumscolopendria, Polypodium scolopendrium, Polypodium scouleri, Polypodiumscutulatum, Polypodium segregatum, Polypodium semihirsutum, Polypodiumsemihirsutum var. fuscosetosum, Polypodium senile var. minor, Polypodiumsericeolanatum, Polypodium serraeforme, Polypodium serricula, Polypodiumsesquipedala, Polypodium sessilifolium, Polypodium setosum var. calvum,Polypodium setulosum, Polypodium shaferi, Polypodium sibomense,Polypodium siccum, Polypodium simacense, Polypodium simulans, Polypodiumsingeri, Polypodium sinicum, Polypodium sintenisii, Polypodium skutchii,Polypodium sloanei, Polypodium sodiroi, Polypodium sordidulum,Polypodium sordidum, Polypodium sphaeropteroides, Polypodium sphenodes,Polypodium sprucei, Polypodium sprucei var. furcativenosa, Polypodiumsteirolepis, Polypodium stenobasis, Polypodium stenolepis, Polypodiumstenopterum, Polypodium subcapillare, Polypodium subflabelliforme,Polypodium subhemionitidium, Polypodium subinaequale, Polypodiumsubintegrum, Polypodium subspathulatum, Polypodium subtile, Polypodiumsubvestitum, Polypodium subviride, Polypodium superficiale var.attenuatum, Polypodium superficiale var. chinensis, Polypodiumsursumcurrens, Polypodium tablazianum, Polypodium taenifolium,Polypodium tamandarei, Polypodium tatei, Polypodium tenuiculum var.acrosora, Polypodium tenuiculum var. brasiliense, Polypodium tenuilore,Polypodium tenuinerve, Polypodium tepuiense, Polypodium teresae,Polypodium tetragonum var. incompletum, Polypodium thysanolepis var.bipinnatifidum, Polypodium thyssanolepis, var. thyssanolepis, Polypodiumthyssanolepsi, Polypodium tobagense, Polypodium trichophyllum,Polypodium tridactylum, Polypodium tridentatum, Polypodium trifurcatumvar. brevipes, Polypodium triglossum, Polypodium truncatulum, Polypodiumtruncicola var. major, Polypodium truncicola var. minor, Polypodiumtuberosum, Polypodium tunguraguae, Polypodium turquinum, Polypodiumturrialbae, Polypodium ursipes, Polypodium vagans, Polypodiumvaldealatum, Polypodium versteegii, Polypodium villagranii, Polypodiumvirginianum f. cambroideum, Polypodium virginianum f. peraferens,Polypodium vittarioides, Polypodium vulgare, Polypodium vulgare L.,Polypodium vulgare subsp. oreophilum, Polypodium vulgare var.acuminatum, Polypodium vulpinum, Polypodium williamsii, Polypodiumwobbense, Polypodium×fallacissimum-guttatum, Polypodium xantholepis,Polypodium xiphopteris, Polypodium yarumalense, Polypodium yungense, andPolypodium zosteriforme.

In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Order Polypodiales, Family Polypodiaceae, GenusPlatycerium.

In some embodiments the PtIP-83 polypeptide is derived from a species inthe Division Lycophyta.

In some embodiments the PtIP-83 polypeptide is derived from a species inthe Class Isoetopsida or Class Lycopodiopsida.

In some embodiments the PtIP-83 polypeptide is derived from a species inthe Class Isoetopsida Order Selaginales. In some embodiments the PtIP-83polypeptide is derived from a fern species in the Class Isoetopsida,Order Selaginales, Family Selaginellaceae. In some embodiments thePtIP-83 polypeptide is derived from a species in the Genus Selaginella.In some embodiments the PtIP-83 polypeptide is derived from a species inthe Class Lycopodiopsida, Order Lycopodiales.

In some embodiments the PtIP-83 polypeptide is derived from a fernspecies in the Class Lycopodiopsida, Order Lycopodiales FamilyLycopodiaceae or Family Huperziaceae.

In some embodiments the PtIP-83 polypeptide is derived from a species inthe Genus Austrolycopodium, Dendrolycopodium, Diphasiastrum, Diphasium,Huperzia, Lateristachys, Lycopodiastrum, Lycopodiella, Lycopodium,Palhinhaea, Pseudodiphasium, Pseudolycopodiella, Pseudolycopodium orSpinulum.

In some embodiments the PtIP-83 polypeptide is derived from a species inthe Genus Lycopodium.

In some embodiments the PtIP-83 polypeptide is derived from a species inthe Genus Huperzia.

Phylogenetic, Sequence Motif, and Structural Analyses for InsecticidalProtein Families

The sequence and structure analysis method employed is composed of fourcomponents: phylogenetic tree construction, protein sequence motifsfinding, secondary structure prediction, and alignment of proteinsequences and secondary structures. Details about each component areillustrated below.

Phylogenetic Tree Construction

The phylogenetic analysis was performed using the software MEGA5.Protein sequences were subjected to ClustalW version 2 analysis (LarkinM. A et al (2007) Bioinformatics 23(21): 2947-2948) for multiplesequence alignment. The evolutionary history was then inferred by theMaximum Likelihood method based on the JTT matrix-based model. The treewith the highest log likelihood was obtained, exported in Newick format,and further processed to extract the sequence IDs in the same order asthey appeared in the tree. A few clades representing sub-families weremanually identified for each insecticidal protein family.

Protein Sequence Motifs Finding

Protein sequences were re-ordered according to the phylogenetic treebuilt previously, and fed to the MOTIF analysis tool MEME (Multiple EMfor MOTIF Elicitation) (Bailey T. L., and Elkan C., Proceedings of theSecond International Conference on Intelligent Systems for MolecularBiology, pp. 28-36, AAAI Press, Menlo Park, Calif., 1994.) foridentification of key sequence motifs. MEME was setup as follows:Minimum number of sites 2, Minimum motif width 5, and Maximum number ofmotifs 30. Sequence motifs unique to each sub-family were identified byvisual observation. The distribution of MOTIFs across the entire genefamily could be visualized in HTML webpage. The MOTIFs are numberedrelative to the ranking of the E-value for each MOTIF. The amino acidsequence MOTIFs identified for each of the PtIP-83 polypeptides and theresidue ranges defining the MOTIFs relative to each of the correspondingsequence identifier (SEQ ID NO:) are shown in Table 2. FIG. 2 shows analignment of the PtIP-83 polypeptides PtIP-83Aa (SEQ ID NO: 1),PtIP-83Ca (SEQ ID NO: 5), PtIP-83Cb (SEQ ID NO: 7), PtIP-83Cc (SEQ IDNO: 9), PtIP-83Cd (SEQ ID NO: 11), PtIP-83Ce (SEQ ID NO: 13), PtIP-83Cf(SEQ ID NO: 15), and PtIP-83Fa (SEQ ID NO: 3), and the location relativeto PtIP-83Aa (SEQ ID NO: 1) of the amino acid sequence MOTIFs present inPtIP-83Aa (SEQ ID NO: 1).

Secondary Structure Prediction

PSIPRED, top ranked secondary structure prediction method (Jones D T.(1999) J. Mol. Biol. 292: 195-202), was installed in local Linux server,and used for protein secondary structure prediction. The tool providesaccurate structure prediction using two feed-forward neural networksbased on the PSI-BLAST output. The PSI-BLAST database was created byremoving low-complexity, transmembrane, and coiled-coil regions inUniref100. The PSIPRED results contain the PtIP-secondary structures(Alpha helix: H, Beta strand: E, and Coil: C) and the correspondingconfidence scores for each amino acid in a given protein sequence. FIG.2 shows the PtIP-83 polypeptide amino acid sequence alignments and theconserved secondary structural regions.

TABLE 2 SEQ ID NO: MOTIF 19 MOTIF 7 MOTIF 13 MOTIF 20 MOTIF 10 MOTIF 18PtIP-83Aa 1  4-17 21-64  71-100 102-120 n.p. n.p. PtIP-83Ca 5  1-1417-60 68-97  98-116 122-171 173-208 PtIP-83Cb 7  1-14 17-60 69-98 n.p.121-170 n.p. PtIP-83Cc 9  1-14 17-60 68-97  98-116 122-171 173-208PtIP-83Cd 11  1-14 17-60 68-97  98-116 122-171 173-208 PtIP-83Ce 13 1-14 17-60 68-97  98-116 121-170 173-208 PtIP-83Cf 15  1-14 21-64 68-97 98-116 122-171 n.p. PtIP-83Fa 3  5-18 17-60 68-97 n.p. n.p. n.p. SEQ IDNO: MOTIF 24 MOTIF 14 MOTIF 11 MOTIF 22 MOTIF 2 MOTIF 8 PtIP-83Aa 1 n.p.238-263 n.p. 313-326 327-376 376-425 PtIP-83Ca 5 211-220 221-246 248-297298-311 312-361 362-411 PtIP-83Cb 7 n.p. 223-248 249-298 299-312 313-362363-412 PtIP-83Cc 9 211-220 221-246 248-297 298-311 312-361 362-411PtIP-83Cd 11 211-220 221-246 248-297 298-311 312-361 362-411 PtIP-83Ce13 211-220 221-246 248-297 298-311 312-361 362-411 PtIP-83Cf 15 211-220221-246 248-297 298-311 312-361 362-411 PtIP-83Fa 3 247-256 262-287 n.p.329-342 344-393 394-443 SEQ ID NO: MOTIF 21 MOTIF 15 MOTIF 9 MOTIF 1MOTIF 17 MOTIF 6 PtIP-83Aa 1 508-521 428-453 455-504 523-572 576-594596-645 PtIP-83Ca 5 493-506 413-438 441-490 508-557 561-579 581-630PtIP-83Cb 7 494-507 414-439 442-491 509-558 562-580 582-631 PtIP-83Cc 9493-506 413-438 441-490 508-557 561-579 581-630 PtIP-83Cd 11 493-506413-438 441-490 508-557 561-579 581-630 PtIP-83Ce 13 493-506 413-438441-490 508-557 561-579 581-630 PtIP-83Cf 15 493-506 413-438 441-490508-557 561-579 581-630 PtIP-83Fa 3 525-538 445-470 473-522 540-589593-611 613-662 SEQ ID NO: MOTIF 12 MOTIF 4 MOTIF 16 MOTIF 5 MOTIF 23MOTIF 3 PtIP-83Aa 1 648-683 684-719 723-741 746-795 798-807 809-858PtIP-83Ca 5 634-669 670-705 708-726 732-781 784-793 795-844 PtIP-83Cb 7635-670 671-706 709-727 731-780 783-792 794-843 PtIP-83Cc 9 634-669670-705 708-726 730-779 782-791 793-842 PtIP-83Cd 11 634-669 670-705708-726 730-779 782-791 793-842 PtIP-83Ce 13 634-669 670-705 708-726730-779 782-791 793-842 PtIP-83Cf 15 634-669 670-705 708-726 731-780783-792 794-843 PtIP-83Fa 3 667-702 703-738 740-758 763-812 815-824826-875 n.p. = not presentAlignment of Protein Sequences and Secondary Structures

A customized script was developed to generate gapped secondary structurealignment according to the multiple protein sequence alignment from step1 for all proteins. All aligned protein sequences and structures wereconcatenated into a single FASTA file, and then imported into MEGA forvisualization and identification of conserved structures.

In some embodiments a PtIP-83 polypeptide comprises an amino acidsequence MOTIF selected from: an amino acid sequence MOTIF 1 asrepresented by an amino acid sequence of the formulaMP[DE]MPSEADWSIFVNE[IV]EAVAEGMPTEVSEVP[AV]WKAKCKN[MV]AALGREM[SC]I (SEQID NO: 646); an amino acid sequence MOTIF 2 as represented by an aminoacid sequence of the formulaPQLQYRMYG[NS]LI[KN]QMAQVAQNYDQ[ED]FKQ[FL]KLFI[IA]QNQI[LF]GSYLLQQN[KR]A F(SEQ ID NO: 647); an amino acid sequence MOTIF 3 as represented by anamino acid sequence of the formulaNTFMQMTPFTRWRLRLSASASENA[EG]LAFPTATA[PL]DSTT[EQ][IV]VITFHVTAIR (SEQ IDNO: 648); an amino acid sequence MOTIF 4 as represented by an amino acidsequence of the formula [DN]FTSRHVVK[GD]IPVSLLLDGEDWEFEIPVQ[AG]GMSSFP(SEQ ID NO: 649); an amino acid sequence MOTIF 5 as represented by anamino acid sequence of the formulaIIHQP[SA]T[RQ][ST]G[IT]VYILLQGSTIFHDRRR[DE]EVMTFQAA[DA]PLN[FY][QH]YAYRLDTG (SEQ ID NO: 650); an amino acid sequence MOTIF 6 as represented by anamino acid sequence of the formulaS[HQ]ADRLAAIQP[AV]DLTN[HY]LEMAT[HQ]MDMRTT[RS][MI]L[IL]GLLN[MI]LRIQNAALMYEY (SEQ ID NO: 651); an amino acid sequence MOTIF 7 as represented by anamino acid sequence of the formula[VL]DRVEFSEVMVIHRMYVRL[SA]DL[ND]VGEL[PE]GA[EG][RK]VKR[VL]YV[FL]ADVVE(SEQ ID NO: 652); an amino acid sequence MOTIF 8 as represented by anamino acid sequence of the formulaA[DE]RELQMESFHSAVISQRRQEL[ND]TA[IF]AKM[DE]R[LM]SLQMEEE[NS]RAMEQAQKE M(SEQ ID NO: 653); an amino acid sequence MOTIF 9 as represented by anamino acid sequence of the formulaFVTAGATAPGA[AV]ASAGQAVSIAGQAAQ[AG]LRRVVEILE[GQ]LEAVMEVVAA[VI]K (SEQ IDNO: 654); an amino acid sequence MOTIF 10 as represented by an aminoacid sequence of the formulaDGMNWG[IT]YI[YH]GE[KE]V[EQ]RSPLLPSNAILAVWADRC[TI]ITSARHNH[VF]NAPGR[IV]I(SEQ ID NO: 655); an amino acid sequence MOTIF 11 as represented by anamino acid sequence of the formula[KV][VK][CA]RPPSPDM[MV]SAVAEHALWLNDVLLQVVQ[KN]ESQ[LM]QGT[AE]PYNECLAL LGR(SEQ ID NO: 656); an amino acid sequence MOTIF 12 as represented by anamino acid sequence of the formulaPTELT[VA]WPLGMDTV[AG]NLLIAQENAAL[VL]GLIQLGPSS (SEQ ID NO: 657); an aminoacid sequence MOTIF 13 as represented by an amino acid sequence of theformula RDQ[MT][HQ]MPGSVTVI[IV]LCRLLQFP[IT]DGSQA[TA]T (SEQ ID NO: 658);an amino acid sequence MOTIF 14 as represented by an amino acid sequenceof the formula TSIPVEVVTDP[SN]ILLGMQTTV[LH]IAEL (SEQ ID NO: 659); anamino acid sequence MOTIF 15 as represented by an amino acid sequence ofthe formula EGLR[EQ]FQNRQVARA[VL]FAVLKAVA[MQ]I[AG] (SEQ ID NO: 660); anamino acid sequence MOTIF 16 as represented by an amino acid sequence ofthe formula W[TS]RVRIRHLEM[QH]F[AV]QEASG (SEQ ID NO: 661); an amino acidsequence MOTIF 17 as represented by an amino acid sequence of theformula QISELQY[ED]IWVQG[LM][ML]RDIA (SEQ ID NO: 662); an amino acidsequence MOTIF 18 as represented by an amino acid sequence of theformula TFTLGSGVTGITSMHGEPSLDPWNGVSLDSASPTAF (SEQ ID NO: 663); an aminoacid sequence MOTIF 19 as represented by an amino acid sequence of theformula MDYSTLYRDLNQIS (SEQ ID NO: 664); an amino acid sequence MOTIF 20as represented by an amino acid sequence of the formulaLRLPFM[QK]LHARVIEQN[VR]K[SE](SEQ ID NO: 665); an amino acid sequenceMOTIF 21 as represented by an amino acid sequence of the formulaVDSLEQVG[QH][IL]V[GD]AP (SEQ ID NO: 666); an amino acid sequence MOTIF22 as represented by an amino acid sequence of the formula[IV][EQ][CA]VMK[IM]GRF[VG][SL]VV (SEQ ID NO: 667); an amino acidsequence MOTIF 23 as represented by an amino acid sequence of theformula TLTNEPSE[EQ]F (SEQ ID NO: 668); and an amino acid sequence MOTIF24 as represented by an amino acid sequence of the formula LPRQSRNISF(SEQ ID NO: 669).

In some embodiments a PtIP-83 polypeptide comprises an amino acidsequence MOTIF selected from: an amino acid sequence MOTIF 1 having atleast 90% sequence identity to the amino acid sequence as represented bythe formulaMP[DE]MPSEADWSIFVNE[IV]EAVAEGMPTEVSEVP[AV]WKAKCKN[MV]AALGREM[SC]I (SEQID NO: 646); an amino acid sequence MOTIF 2 having at least 90% sequenceidentity to the amino acid sequence as represented by the formulaPQLQYRMYG[NS]LI[KN]QMAQVAQNYDQ[ED]FKQ[FL]KLFI[IA]QNQI[LF]GSYLLQQN[KR]A F(SEQ ID NO: 647); an amino acid sequence MOTIF 3 having at least 90%sequence identity to the amino acid sequence as represented by theformula NTFMQMTPFTRWRLRLSASASENA[EG]LAFPTATA[PL]DSTT[EQ][IV]VITFHVTAIR(SEQ ID NO: 648); an amino acid sequence MOTIF 4 having at least 90%sequence identity to the amino acid sequence as represented by theformula [DN]FTSRHVVK[GD]IPVSLLLDGEDWEFEIPVQ[AG]GMSSFP (SEQ ID NO: 649);an amino acid sequence MOTIF 5 having at least 90% sequence identity tothe amino acid sequence as represented by the formulaIIHQP[SA]T[RQ][ST]G[IT]VYILLQGSTIFHDRRR[DE]EVMTFQAA[DA]PLN[FY][QH]YAYRLDTG (SEQ ID NO: 650); an amino acid sequence MOTIF 6 having at least 90%sequence identity to the amino acid sequence as represented by theformulaS[HQ]ADRLAAIQP[AV]DLTN[HY]LEMAT[HQ]MDMRTT[RS][MI]L[IL]GLLN[MI]LRIQNAALMYEY (SEQ ID NO: 651); an amino acid sequence MOTIF 7 having at least 90%sequence identity to the amino acid sequence as represented by theformula[VL]DRVEFSEVMVIHRMYVRL[SA]DL[ND]VGEL[PE]GA[EG][RK]VKR[VL]YV[FL]ADVVE(SEQ ID NO: 652); an amino acid sequence MOTIF 8 having at least 90%sequence identity to the amino acid sequence as represented by theformulaA[DE]RELQMESFHSAVISQRRQEL[ND]TA[IF]AKM[DE]R[LM]SLQMEEE[NS]RAMEQAQKE M(SEQ ID NO: 653); an amino acid sequence MOTIF 9 having at least 90%sequence identity to the amino acid sequence as represented by theformula FVTAGATAPGA[AV]ASAGQAVSIAGQAAQ[AG]LRRVVEILE[GQ]LEAVMEVVAA[VI]K(SEQ ID NO: 654); an amino acid sequence MOTIF 10 having at least 90%sequence identity to the amino acid sequence as represented by theformulaDGMNWG[IT]YI[YH]GE[KE]V[EQ]RSPLLPSNAILAVWADRC[TI]ITSARHNH[VF]NAPGR[IV]I(SEQ ID NO: 655); an amino acid sequence MOTIF 11 having at least 90%sequence identity to the amino acid sequence as represented by theformula[KV][VK][CA]RPPSPDM[MV]SAVAEHALWLNDVLLQVVQ[KN]ESQ[LM]QGT[AE]PYNECLAL LGR(SEQ ID NO: 656); an amino acid sequence MOTIF 12 having at least 90%sequence identity to the amino acid sequence as represented by theformula PTELT[VA]WPLGMDTV[AG]NLLIAQENAAL[VL]GLIQLGPSS (SEQ ID NO: 657);an amino acid sequence MOTIF 13 having at least 90% sequence identity tothe amino acid sequence as represented by the formulaRDQ[MT][HQ]MPGSVTVI[IV]LCRLLQFP[IT]DGSQA[TA]T (SEQ ID NO: 658); an aminoacid sequence MOTIF 14 having at least 90% sequence identity to theamino acid sequence as represented by the formulaTSIPVEVVTDP[SN]ILLGMQTTV[LH]IAEL (SEQ ID NO: 659); an amino acidsequence MOTIF 15 having at least 90% sequence identity to the aminoacid sequence as represented by the formulaEGLR[EQ]FQNRQVARA[VL]FAVLKAVA[MQ]I[AG] (SEQ ID NO: 660); an amino acidsequence MOTIF 16 having at least 90% sequence identity to the aminoacid sequence as represented by the formula W[TS]RVRIRHLEM[QH]F[AV]QEASG(SEQ ID NO: 661); an amino acid sequence MOTIF 17 having at least 90%sequence identity to the amino acid sequence as represented by theformula QISELQY[ED]IWVQG[LM][ML]RDIA (SEQ ID NO: 662); an amino acidsequence MOTIF 18 having at least 90% sequence identity to the aminoacid sequence as represented by the formulaTFTLGSGVTGITSMHGEPSLDPWNGVSLDSASPTAF (SEQ ID NO: 663); an amino acidsequence MOTIF 19 having at least 90% sequence identity to the aminoacid sequence as represented by the formula MDYSTLYRDLNQIS (SEQ ID NO:664); an amino acid sequence MOTIF 20 having at least 90% sequenceidentity to the amino acid sequence as represented by the formulaLRLPFM[QK]LHARVIEQN[VR]K[SE] (SEQ ID NO: 665); an amino acid sequenceMOTIF 21 having at least 90% sequence identity to the amino acidsequence as represented by the formula VDSLEQVG[QH][IL]V[GD]AP (SEQ IDNO: 666); an amino acid sequence MOTIF 22 having at least 90% sequenceidentity to the amino acid sequence as represented by the formula[IV][EQ][CA]VMK[IM]GRF[VG][SL]VV (SEQ ID NO: 667); an amino acidsequence MOTIF 23 having at least 90% sequence identity to the aminoacid sequence as represented by the formula TLTNEPSE[EQ]F (SEQ ID NO:668); and an amino acid sequence MOTIF 24 having at least 90% sequenceidentity to the amino acid sequence as represented by the formulaLPRQSRNISF (SEQ ID NO: 669).

In some embodiments a PtIP-83 polypeptide comprises an amino acidsequence MOTIF selected from: an amino acid sequence MOTIF 1 asrepresented by an amino acid sequence of the formulaMP[DE]MP[ST][ED]ADWSIFVNE[IVL]EAVAEGMPTEVSEVP[AV]W[KR]AKCKN[MV]AALGREM[SC]I (SEQ ID NO: 670); an amino acid sequence MOTIF 2 as representedby an amino acid sequence of the formulaPQLQYRMYG[NS]LI[KRN]QMAQVAQNYD[QR][ED]FK[QR][FL][KR]LFI[IAVL]QNQI[LF]GSYLL[QE]QN[KR]AF (SEQ ID NO: 671); an amino acid sequence MOTIF 3 asrepresented by an amino acid sequence of the formulaN[TK]FMQMTPFT[RH]WRLRLSASA[SPKA]EN[AK][EG]LAFPTATA[PL]DSTT[EQ][IV][VA]ITFHVTAIR (SEQ ID NO: 672); an amino acid sequence MOTIF 4 as representedby an amino acid sequence of the formula[DN]FTSRHVVK[GD]IPV[SN]LLLDG[EG]DWEFEIPVQ[AG]GMSSFP (SEQ ID NO: 673); anamino acid sequence MOTIF 5 as represented by an amino acid sequence ofthe formulaIIHQP[SA]T[RQ][ST]G[IT][VI]YILLQGST[IV]FHDRRR[DE][EQ]V[ML]T[FP]QAA[DAV]PLN[FY][QH]YAYRLDTG(SEQ ID NO: 674); an amino acid sequence MOTIF 6 as represented by anamino acid sequence of the formulaS[HQ]ADRLAAIQP[AV][DN]LTN[HYF]LEMAT[HQ]MDMRTT[RS][MI]L[IL]GLLN[MI][LM]RIQNAAL[MR]YEY (SEQ ID NO: 675); an amino acid sequence MOTIF 7 asrepresented by an amino acid sequence of the formula[VL]D[RQ]VEFSEVMVIHRMYV[N]RL[SA]DL[ND]V[GA][EQ]L[PE]GA[EG][RK]VKR[VL]YV[FL]ADVVE(SEQ ID NO: 676); an amino acid sequence MOTIF 8 as represented by anamino acid sequence of the formulaA[DE]RELQMESFH[SA]AVISQ[RK]R[QGE]EL[ND][TD][AT][IF]AKM[DE]R[LM]SLQMEEE[NSD][RG]AMEQA[QR]KEM (SEQ ID NO: 677); an amino acid sequence MOTIF 9 asrepresented by an amino acid sequence of the formulaF[VL]TAGATAPGA[AV]ASAGQAV[SN]IAGQAAQ[AG]LRRVVEILE[GQ]LEAVMEVVAA[VI]K(SEQ ID NO: 678); an amino acid sequence MOTIF 10 as represented by anamino acid sequence of the formulaD[GD][MA][NK]WG[IT]Y[IV][YH][GA]E[KE]V[EQ][RVL]SPL[LYF][PN][SNG][NW][ASP][IY]L[AGV]V[WE]A[DQ]R[CS][TI]IT[SA]A[RFM]HN[HVT][VF][ND][AER]PG[RW][IV][IR] (SEQID NO: 679); an amino acid sequence MOTIF 11 as represented by an aminoacid sequence of the formula[KV][VK][CA][RGC][PHY]PSP[DE][MIL][MV]SAV[AG][EV]HA[LIN]WL[NS][DK]VLL[QR]VVQ[KN]ES[QH][LM]QGT[AE][PSA]YNECLALLGR (SEQ ID NO: 680); an amino acidsequence MOTIF 12 as represented by an amino acid sequence of theformula[PN]T[EQ]LT[VAT]WPL[GR]MDTV[AG][ND]LLI[AT][QH]E[NS]AAL[VLS]GL[ITMA]QLG[PQ][SP]S (SEQ ID NO: 681); an amino acid sequence MOTIF 13 as represented byan amino acid sequence of the formula[RLC][DLWK][QNPR][MTP][HQR][MIL]PGSVTVI[IV]LCRLLQFP[IT][DG]G[SR][QFR][AS][TAD][TW](SEQ ID NO: 682); an amino acid sequence MOTIF 14 as represented by anamino acid sequence of the formula[TA][SGV][IL]PV[ED]VVTDP[SN]IL[LM]GMQT[TS]V[LH]IAEL (SEQ ID NO: 683); anamino acid sequence MOTIF 15 as represented by an amino acid sequence ofthe formula EGLR[EQ]FQN[RE]QVA[RN]A[VL]FAVL[KS][AS]VA[MQ]I[AG] (SEQ IDNO: 684); an amino acid sequence MOTIF 16 as represented by an aminoacid sequence of the formula W[TS]RVRIRHLEM[QH]F[AV][QK]E[AS][SM][GN](SEQ ID NO: 685); an amino acid sequence MOTIF 17 as represented by anamino acid sequence of the formula Q[IM]S[EQ]LQY[ED]IWVQG[LM][ML]RD[IM]A(SEQ ID NO: 686); an amino acid sequence MOTIF 18 as represented by anamino acid sequence of the formula TFTLGSGVTGITSMHGEPSLDPWNGVSLDSASPTAF(SEQ ID NO: 663); an amino acid sequence MOTIF 19 as represented by anamino acid sequence of the formula [MLV]DY[SK][TSK]L[YF][RE]DLNQIS (SEQID NO: 687); an amino acid sequence MOTIF 20 as represented by an aminoacid sequence of the formulaL[RHQ]L[PT]FM[QK]LHA[RIT][VQL][IR]E[QER][NF][VR][KWS][SE] (SEQ ID NO:688); an amino acid sequence MOTIF 21 as represented by an amino acidsequence of the formula V[DN][SA]L[ED]QV[GS][QH][IL]V[GD]AP (SEQ ID NO:689); an amino acid sequence MOTIF 22 as represented by an amino acidsequence of the formula[IV][EQH][CAS][VA][MI]K[IM][GV][RP][FI][VG][SL]VV (SEQ ID NO: 690); anamino acid sequence MOTIF 23 as represented by an amino acid sequence ofthe formula TLTN[EQ]PSE[EQDH]F (SEQ ID NO: 691); and an amino acidsequence MOTIF 24 as represented by an amino acid sequence of theformula LP[RS]QS[RT]N[IV]SF (SEQ ID NO: 692).

In some embodiments a PtIP-83 polypeptide comprises an amino acidsequence MOTIF selected from: an amino acid sequence MOTIF 1 having atleast 90% sequence identity to the amino acid sequence as represented bythe formulaMP[DE]MP[ST][ED]ADWSIFVNE[IVL]EAVAEGMPTEVSEVP[AV]W[KR]AKCKN[MV]AALGREM[SC]I (SEQ ID NO: 670); an amino acid sequence MOTIF 2 having at least90% sequence identity to the amino acid sequence as represented by theformulaPQLQYRMYG[NS]LI[KRN]QMAQVAQNYD[QR][ED]FK[QR][FL][KR]LFI[IAVL]QNQI[LF]GSYLL[QE]QN[KR]AF (SEQ ID NO: 671); an amino acid sequence MOTIF 3 having atleast 90% sequence identity to the amino acid sequence as represented bythe formulaN[TK]FMQMTPFT[RH]WRLRLSASA[SPKA]EN[AK][EG]LAFPTATA[PL]DSTT[EQ][IV][VA]ITFHVTAIR (SEQ ID NO: 672); an amino acid sequence MOTIF 4 having at least90% sequence identity to the amino acid sequence as represented by theformula [DN]FTSRHVVK[GD]IPV[SN]LLLDG[EG]DWEFEIPVQ[AG]GMSSFP (SEQ ID NO:673); an amino acid sequence MOTIF 5 having at least 90% sequenceidentity to the amino acid sequence as represented by the formulaIIHQP[SA]T[RQ][ST]G[IT][VI]YILLQGST[IV]FHDRRR[DE][EQ]V[ML]T[FP]QAA[DAV]PLN[FY][QH]YAYRLDTG(SEQ ID NO: 674); an amino acid sequence MOTIF 6 having at least 90%sequence identity to the amino acid sequence as represented by theformulaS[HQ]ADRLAAIQP[AV][DN]LTN[HYF]LEMAT[HQ]MDMRTT[RS][MI]L[IL]GLLN[MI][LM]RIQNAAL[MR]YEY (SEQ ID NO: 675); an amino acid sequence MOTIF 7 having atleast 90% sequence identity to the amino acid sequence as represented bythe formula[VL]D[RQ]VEFSEVMVIHRMYV[N]RL[SA]DL[ND]V[GA][EQ]L[PE]GA[EG][RK]VKR[VL]YV[FL]ADVVE(SEQ ID NO: 676); an amino acid sequence MOTIF 8 having at least 90%sequence identity to the amino acid sequence as represented by theformulaA[DE]RELQMESFH[SA]AVISQ[RK]R[QGE]EL[ND][TD][AT][IF]AKM[DE]R[LM]SLQMEEE[NSD][RG]AMEQA[QR]KEM (SEQ ID NO: 677); an amino acid sequence MOTIF 9having at least 90% sequence identity to the amino acid sequence asrepresented by the formulaF[VL]TAGATAPGA[AV]ASAGQAV[SN]IAGQAAQ[AG]LRRVVEILE[GQ]LEAVMEVVAA[VI]K(SEQ ID NO: 678); an amino acid sequence MOTIF 10 having at least 90%sequence identity to the amino acid sequence as represented by theformulaD[GD][MA][NK]WG[IT]Y[IV][YH][GA]E[KE]V[EQ][RVL]SPL[LYF][PN][SNG][NW][ASP][IY]L[AGV]V[WE]A[DQ]R[CS][TI]IT[SA]A[RFM]HN[HVT][VF][ND][AER]PG[RW][IV][IR] (SEQID NO: 679); an amino acid sequence MOTIF 11 having at least 90%sequence identity to the amino acid sequence as represented by theformula[KV][VK][CA][RGC][PHY]PSP[DE][MIL][MV]SAV[AG][EV]HA[LIN]WL[NS][DK]VLL[QR]VVQ[KN]ES[QH][LM]QGT[AE][PSA]YNECLALLGR (SEQ ID NO: 680); an amino acidsequence MOTIF 12 having at least 90% sequence identity to the aminoacid sequence as represented by the formula[PN]T[EQ]LT[VAT]WPL[GR]MDTV[AG][ND]LLI[AT][QH]E[NS]AAL[VLS]GL[ITMA]QLG[PQ][SP]S (SEQ ID NO: 681); an amino acid sequence MOTIF 13 having at least90% sequence identity to the amino acid sequence as represented by theformula[RLC][DLWK][QNPR][MTP][HQR][MIL]PGSVTVI[IV]LCRLLQFP[IT][DG]G[SR][QFR][AS][TAD][TW](SEQ ID NO: 682); an amino acid sequence MOTIF 14 having at least 90%sequence identity to the amino acid sequence as represented by theformula [TA][SGV][IL]PV[ED]VVTDP[SN]IL[LM]GMQT[TS]V[LH]IAEL (SEQ ID NO:683); an amino acid sequence MOTIF 15 having at least 90% sequenceidentity to the amino acid sequence as represented by the formulaEGLR[EQ]FQN[RE]QVA[RN]A[VL]FAVL[KS][AS]VA[MQ]I[AG](SEQ ID NO: 684); anamino acid sequence MOTIF 16 having at least 90% sequence identity tothe amino acid sequence as represented by the formulaW[TS]RVRIRHLEM[QH]F[AV][QK]E[AS][SM][GN] (SEQ ID NO: 685); an amino acidsequence MOTIF 17 having at least 90% sequence identity to the aminoacid sequence as represented by the formulaQ[IM]S[EQ]LQY[ED]IWVQG[LM][ML]RD[IM]A (SEQ ID NO: 686); an amino acidsequence MOTIF 18 having at least 90% sequence identity to the aminoacid sequence as represented by the formulaTFTLGSGVTGITSMHGEPSLDPWNGVSLDSASPTAF (SEQ ID NO: 663); an amino acidsequence MOTIF 19 having at least 90% sequence identity to the aminoacid sequence as represented by the formula[MLV]DY[SK][TSK]L[YF][RE]DLNQIS (SEQ ID NO: 687); an amino acid sequenceMOTIF 20 having at least 90% sequence identity to the amino acidsequence as represented by the formulaL[RHQ]L[PT]FM[QK]LHA[RIT][VQL][IR]E[QER][NF][VR][KWS][SE] (SEQ ID NO:688); an amino acid sequence MOTIF 21 having at least 90% sequenceidentity to the amino acid sequence as represented by the formulaV[DN][SA]L[ED]QV[GS][QH][IL]V[GD]AP (SEQ ID NO: 689); an amino acidsequence MOTIF 22 having at least 90% sequence identity to the aminoacid sequence as represented by the formula[IV][EQH][CAS][VA][MI]K[IM][GV][RP][FI][VG][SL]VV (SEQ ID NO: 690); anamino acid sequence MOTIF 23 having at least 90% sequence identity tothe amino acid sequence as represented by the formula TLTN[EQ]PSE[EQDH]F(SEQ ID NO: 691); and an amino acid sequence MOTIF 24 having at least90% sequence identity to the amino acid sequence as represented by theformula LP[RS]QS[RT]N[IV]SF (SEQ ID NO: 692).

In some embodiments a PtIP-83 polypeptide comprises an amino acidsequence MOTIF selected from: an amino acid sequence MOTIF 1 asrepresented by an amino acid sequence of the formulaMP[DE]MP[ST][ED]ADWSIFVNE[IVL]EAVAEGMPTEVSEVP[AVIL]W[KR]AKCKN[MVIL]AALGREM[SCT]I (SEQ ID NO: 693); an amino acid sequence MOTIF 2 asrepresented by an amino acid sequence of the formulaPQLQYRMYG[NS]LI[KRNQ]QMAQVAQNYD[QRNK][ED]FK[QRNK][FL][KR]LFI[IAVL]QNQI[LFIV]GSYLL[QEND]QN[KR]AF (SEQ ID NO: 694); an amino acid sequence MOTIF 3as represented by an amino acid sequence of the formulaN[TKSR]FMQMTPFT[RHK]WRLRLSASA[SPKATR]EN[AKR][EG]LAFPTATA[PLIV]DSTT[EQND][IVL][VAIL]ITFHVTAIR (SEQ ID NO: 695); an amino acid sequence MOTIF 4as represented by an amino acid sequence of the formula[DNQE]FTSRHVVK[GDE]IPV[SNTQ]LLLDG[EGD]DWEFEIPVQ[AG]GMSSFP (SEQ ID NO:696); an amino acid sequence MOTIF 5 as represented by an amino acidsequence of the formulaIIHQP[SAT]T[RQKN][ST]G[ITLVS][VIL]YILLQGST[IVL]FHDRRR[DE][EQDN]V[MLIV]T[FP]QAA[DAVEIL]PLN[FY][QHN]YAYRLDTG (SEQ ID NO: 697); an amino acid sequenceMOTIF 6 as represented by an amino acid sequence of the formulaS[HQN]ADRLAAIQP[AVIL][DN]LTN[HYF]LEMAT[HQN]MDMRTT[RSKT][MILV]L[ILV]GLLN[MILV][LMIV]RIQNAAL[MRILVK]YEY (SEQ ID NO: 698); an amino acid sequenceMOTIF 7 as represented by an amino acid sequence of the formula[VLI]D[RQKN]VEFSEVMVIHRMYV[N]RL[SAT]DL[NDQE]V[GA][EQND]L[PED]GA[EGD][RK]VKR[VLI]YV[FLIV]ADVVE(SEQ ID NO: 699); an amino acid sequence MOTIF 8 as represented by anamino acid sequence of the formulaA[DE]RELQMESFH[SAT]AVISQ[RK]R[QGEND]EL[NDQE][TDSE][ATS][IFLV]AKM[DE]R[LMIV]SLQMEEE[NSDQET][RGK]AMEQA[QRNK]KEM (SEQ ID NO: 700); an amino acidsequence MOTIF 9 as represented by an amino acid sequence of the formulaF[VLI]TAGATAPGA[AVIL]ASAGQAV[SNTQ]IAGQAAQ[AG]LRRVVEILE[GQN]LEAVMEVVAA[VIL]K (SEQ ID NO: 701); an amino acid sequence MOTIF 10 as representedby an amino acid sequence of the formulaD[GDE][MA][NKQK]WG[ITLVS]Y[IVL][YH][GA]E[KERD]V[EQND][RVLKI]SPL[LYFIV][PNQ][SNGTQ][NWQ][ASPT][IYLV]L[AGVIL]V[WED]A[DQNE]R[CST][TISLV]IT[SAT]A[RFMK]HN[HVTILS][VFIL][NDQE][AERDK]PG[RWK][IVL][IRLVK] (SEQ ID NO: 702); an aminoacid sequence MOTIF 11 as represented by an amino acid sequence of theformula[KVRIL][VKRIL][CA][RGCK][PHY]PSP[DE][MILV][MVIL]SAV[AG][EVDIL]HA[LINVQ]WL[NSQT][DKER]VLL[QRNK]VVQ[KNRQ]ES[QHN][LMIV]QGT[AED][PSAT]YNECLALLGR (SEQ IDNO: 703); an amino acid sequence MOTIF 12 as represented by an aminoacid sequence of the formula[PNQ]T[EQDN]LT[VATILS]WPL[GRK]MDTV[AG][NDQE]LLI[ATS][QHN]E[NSQT]AAL[VLSIT]GL[ITMALVS]QLG[PQN][SPT]S(SEQ ID NO: 704); an amino acid sequence MOTIF 13 as represented by anamino acid sequence of the formula[RLCKIV][DLWKEIVR][QNPRK][MTP][HQR][MILV]PGSVTVI[IVL]LCRLLQFP[ITLVS][DGE]G[SRTK][QFRNK][AST][TADES][TWS](SEQ ID NO: 705); an amino acid sequence MOTIF 14 as represented by anamino acid sequence of the formula[TA][SGVTIL][ILV]PV[ED]VVTDP[SNTQ]IL[LMIV]GMQT[TS]V[LHIV]IAEL (SEQ IDNO: 706); an amino acid sequence MOTIF 15 as represented by an aminoacid sequence of the formulaEGLR[EQND]FQN[REKD]QVA[RNKQ]A[VLI]FAVL[KSRT][AST]VA[MQN]I[AG] (SEQ IDNO: 707); an amino acid sequence MOTIF 16 as represented by an aminoacid sequence of the formulaW[TS]RVRIRHLEM[QHN]F[AVIL][QKNR]E[AST][SMT][GNQ] (SEQ ID NO: 708); anamino acid sequence MOTIF 17 as represented by an amino acid sequence ofthe formula Q[IMLV]S[EQND]LQY[ED]IWVQG[LMIV][MLIV]RD[IMLV]A (SEQ ID NO:709); an amino acid sequence MOTIF 18 as represented by an amino acidsequence of the formula TFTLGSGVTGITSMHGEPSLDPWNGVSLDSASPTAF (SEQ ID NO:663); an amino acid sequence MOTIF 19 as represented by an amino acidsequence of the formula [MLVI]DY[SKTR][TSKR]L[YF][REKD]DLNQIS (SEQ IDNO: 710); an amino acid sequence MOTIF 20 as represented by an aminoacid sequence of the formulaL[RHQKN]L[PTS]FM[QKNR]LHA[RITKLVS][VQLIN][IRLVK]E[QERNDK][NFQ][VRILK][KWSRT][SETD] (SEQ ID NO: 711); an amino acid sequence MOTIF 21 asrepresented by an amino acid sequence of the formulaV[DNQE][SAT]L[ED]QV[GST][QHN][ILV]V[GDE]AP (SEQ ID NO: 712); an aminoacid sequence MOTIF 22 as represented by an amino acid sequence of theformula[IVL][EQHND][CAST][VAIL][MILV]K[IMLV][GVIL][RPK][FILV][VGIL][SLTIV]VV(SEQ ID NO: 713); an amino acid sequence MOTIF 23 as represented by anamino acid sequence of the formula TLTN[EQDN]PSE[EQDHN]F (SEQ ID NO:714); and an amino acid sequence MOTIF 24 as represented by an aminoacid sequence of the formula LP[RSKT]QS[RTKS]N[IVL]SF (SEQ ID NO: 715).

In some embodiments a PtIP-83 polypeptide comprises an amino acidsequence MOTIF selected from: an amino acid sequence MOTIF 1 having atleast 90% sequence identity to the amino acid sequence as represented bythe formulaMP[DE]MP[ST][ED]ADWSIFVNE[IVL]EAVAEGMPTEVSEVP[AVIL]W[KR]AKCKN[MVIL]AALGREM[SCT]I (SEQ ID NO: 693); an amino acid sequence MOTIF 2 having atleast 90% sequence identity to the amino acid sequence as represented bythe formulaPQLQYRMYG[NS]LI[KRNQ]QMAQVAQNYD[QRNK][ED]FK[QRNK][FL][KR]LFI[IAVL]QNQI[LFIV]GSYLL[QEND]QN[KR]AF (SEQ ID NO: 694); an amino acid sequence MOTIF 3having at least 90% sequence identity to the amino acid sequence asrepresented by the formulaN[TKSR]FMQMTPFT[RHK]WRLRLSASA[SPKATR]EN[AKR][EG]LAFPTATA[PLIV]DSTT[EQND][IVL][VAIL]ITFHVTAIR (SEQ ID NO: 695); an amino acid sequence MOTIF 4having at least 90% sequence identity to the amino acid sequence asrepresented by the formula[DNQE]FTSRHVVK[GDE]IPV[SNTQ]LLLDG[EGD]DWEFEIPVQ[AG]GMSSFP (SEQ ID NO:696); an amino acid sequence MOTIF 5 having at least 90% sequenceidentity to the amino acid sequence as represented by the formulaIIHQP[SAT]T[RQKN][ST]G[ITLVS][VIL]YILLQGST[IVL]FHDRRR[DE][EQDN]V[MLIV]T[FP]QAA[DAVEIL]PLN[FY][QHN]YAYRLDTG (SEQ ID NO: 697); an amino acid sequenceMOTIF 6 having at least 90% sequence identity to the amino acid sequenceas represented by the formulaS[HQN]ADRLAAIQP[AVIL][DN]LTN[HYF]LEMAT[HQN]MDMRTT[RSKT][MILV]L[ILV]GLLN[MILV][LMIV]RIQNAAL[MRILVK]YEY (SEQ ID NO: 698); an amino acid sequenceMOTIF 7 having at least 90% sequence identity to the amino acid sequenceas represented by the formula[VLI]D[RQKN]VEFSEVMVIHRMYV[N]RL[SAT]DL[NDQE]V[GA][EQND]L[PED]GA[EGD][RK]VKR[VLI]YV[FLIV]ADVVE(SEQ ID NO: 699); an amino acid sequence MOTIF 8 having at least 90%sequence identity to the amino acid sequence as represented by theformulaA[DE]RELQMESFH[SAT]AVISQ[RK]R[QGEND]EL[NDQE][TDSE][ATS][IFLV]AKM[DE]R[LMIV]SLQMEEE[NSDQET][RGK]AMEQA[QRNK]KEM (SEQ ID NO: 700); an amino acidsequence MOTIF 9 having at least 90% sequence identity to the amino acidsequence as represented by the formulaF[VLI]TAGATAPGA[AVIL]ASAGQAV[SNTQ]IAGQAAQ[AG]LRRVVE ILE[GQN]LEAVMEVVAA[VIL]K (SEQ ID NO: 701); an amino acid sequence MOTIF 10 having atleast 90% sequence identity to the amino acid sequence as represented bythe formulaD[GDE][MA][NKQK]WG[ITLVS]Y[IVL][YH][GA]E[KERD]V[EQND][RVLKI]SPL[LYFIV][PNQ][SNGTQ][NWQ][ASPT][IYLV]L[AGVIL]V[WED]A[DQNE]R[CST][TISLV]IT[SAT]A[RFMK]HN[HVTILS][VFIL][NDQE][AERDK]PG[RWK][IVL][IRLVK] (SEQ ID NO: 702); an aminoacid sequence MOTIF 11 having at least 90% sequence identity to theamino acid sequence as represented by the formula[KVRIL][VKRIL][CA][RGCK][PHY]PSP[DE][MILV][MVIL]SAV[AG][EVDIL]HA[LINVQ]WL[NSQT][DKER]VLL[QRNK]VVQ[KNRQ]ES[QHN][LMIV]QGT[AED][PSAT]YNECLALLGR (SEQ IDNO: 703); an amino acid sequence MOTIF 12 having at least 90% sequenceidentity to the amino acid sequence as represented by the formula[PNQ]T[EQDN]LT[VATILS]WPL[GRK]MDTV[AG][NDQE]LLI[ATS][QHN]E[NSQT]AAL[VLSIT]GL[ITMALVS]QLG[PQN][SPT]S(SEQ ID NO: 704); an amino acid sequence MOTIF 13 having at least 90%sequence identity to the amino acid sequence as represented by theformula[RLCKIV][DLWKEIVR][QNPRK][MTP][HQR][MILV]PGSVTVI[IVL]LCRLLQFP[ITLVS][DGE]G[SRTK][QFRNK][AST][TADES][TWS](SEQ ID NO: 705); an amino acid sequence MOTIF 14 having at least 90%sequence identity to the amino acid sequence as represented by theformula [TA][SGVTIL][ILV]PV[ED]VVTDP[SNTQ]IL[LMIV]GMQT[TS]V[LHIV]IAEL(SEQ ID NO: 706); an amino acid sequence MOTIF 15 having at least 90%sequence identity to the amino acid sequence as represented by theformula EGLR[EQND]FQN[REKD]QVA[RNKQ]A[VLI]FAVL[KSRT][AST]VA[MQN]I[AG](SEQ ID NO: 707); an amino acid sequence MOTIF 16 having at least 90%sequence identity to the amino acid sequence as represented by theformula W[TS]RVRIRHLEM[QHN]F[AVIL][QKNR]E[AST][SMT][GNQ] (SEQ ID NO:708); an amino acid sequence MOTIF 17 having at least 90% sequenceidentity to the amino acid sequence as represented by the formulaQ[IMLV]S[EQND]LQY[ED]IWVQG[LMIV][MLIV]RD[IMLV]A (SEQ ID NO: 709); anamino acid sequence MOTIF 18 having at least 90% sequence identity tothe amino acid sequence as represented by the formulaTFTLGSGVTGITSMHGEPSLDPWNGVSLDSASPTAF (SEQ ID NO: 663); an amino acidsequence MOTIF 19 having at least 90% sequence identity to the aminoacid sequence as represented by the formula[MLVI]DY[SKTR][TSKR]L[YF][REKD]DLNQIS (SEQ ID NO: 710); an amino acidsequence MOTIF 20 having at least 90% sequence identity to the aminoacid sequence as represented by the formulaL[RHQKN]L[PTS]FM[QKNR]LHA[RITKLVS][VQLIN][IRLVK]E[QERNDK][NFQ][VRILK][KWSRT][SETD] (SEQ ID NO: 711); an amino acid sequence MOTIF 21 having atleast 90% sequence identity to the amino acid sequence as represented bythe formula V[DNQE][SAT]L[ED]QV[GST][QHN][ILV]V[GDE]AP (SEQ ID NO: 712);an amino acid sequence MOTIF 22 having at least 90% sequence identity tothe amino acid sequence as represented by the formula[IVL][EQHND][CAST][VAIL][MILV]K[IMLV][GVIL][RPK][FILV][VGIL][SLTIV]VV(SEQ ID NO: 713); an amino acid sequence MOTIF 23 having at least 90%sequence identity to the amino acid sequence as represented by theformula TLTN[EQDN]PSE[EQDHN]F (SEQ ID NO: 714); and an amino acidsequence MOTIF 24 having at least 90% sequence identity to the aminoacid sequence as represented by the formula LP[RSKT]QS[RTKS]N[IVL]SF(SEQ ID NO: 715).

In some embodiment a PtIP-83 polypeptide comprises, sequentially fromthe N-terminus to the C-terminus, an amino acid sequence MOTIF selectedfrom: MOTIF 19 (SEQ ID NO: 664, SEQ ID NO: 687 or SEQ ID NO: 710), MOTIF7 (SEQ ID NO: 652, SEQ ID NO: 676 or SEQ ID NO: 699), MOTIF 13 (SEQ IDNO: 658, SEQ ID NO: 682 or SEQ ID NO: 705), MOTIF 20 (SEQ ID NO: 665,SEQ ID NO: 688 or SEQ ID NO: 711), MOTIF 10 (SEQ ID NO: 655, SEQ ID NO:679 or SEQ ID NO: 702), MOTIF 18 (SEQ ID NO: 663), MOTIF 24 (SEQ ID NO:669, SEQ ID NO: 692 or SEQ ID NO: 715), MOTIF 14 (SEQ ID NO: 659, SEQ IDNO: 683 or SEQ ID NO: 706), MOTIF 11 (SEQ ID NO: 656, SEQ ID NO: 680 orSEQ ID NO: 703), MOTIF 22 (SEQ ID NO: 667, SEQ ID NO: 690 or SEQ ID NO:713), MOTIF 2 (SEQ ID NO: 647, SEQ ID NO: 671 or SEQ ID NO: 694), MOTIF8 (SEQ ID NO: 653, SEQ ID NO: 677 or SEQ ID NO: 700), MOTIF 15 (SEQ IDNO: 660, SEQ ID NO: 684 or SEQ ID NO: 707), MOTIF 9 (SEQ ID NO: 654, SEQID NO: 678 or SEQ ID NO: 701), MOTIF 21 (SEQ ID NO: 666, SEQ ID NO: 689or SEQ ID NO: 712), MOTIF 1 (SEQ ID NO: 646, SEQ ID NO: 670 or SEQ IDNO: 693), MOTIF 17 (SEQ ID NO: 662, SEQ ID NO: 686 or SEQ ID NO: 709),MOTIF 6 (SEQ ID NO: 651, SEQ ID NO: 675 or SEQ ID NO: 698), MOTIF 12(SEQ ID NO: 657, SEQ ID NO: 681 or SEQ ID NO: 704), MOTIF 4 (SEQ ID NO:649, SEQ ID NO: 673 or SEQ ID NO: 696), MOTIF 16 (SEQ ID NO: 661, SEQ IDNO: 685 or SEQ ID NO: 708), MOTIF 5 (SEQ ID NO: 650, SEQ ID NO: 674 orSEQ ID NO: 697), MOTIF 23 (SEQ ID NO: 668, SEQ ID NO: 691 or SEQ ID NO:714), and MOTIF 3 (SEQ ID NO: 648, SEQ ID NO: 672 or SEQ ID NO: 695).

In some embodiments a PtIP-83 polypeptide comprises, sequentially fromthe N-terminus to the C-terminus, an amino acid sequence MOTIF selectedfrom: MOTIF 19 (SEQ ID NO: 664, SEQ ID NO: 687 or SEQ ID NO: 710), MOTIF7 (SEQ ID NO: 652, SEQ ID NO: 676 or SEQ ID NO: 699), MOTIF 13 (SEQ IDNO: 658, SEQ ID NO: 682 or SEQ ID NO: 705), MOTIF 20 (SEQ ID NO: 665,SEQ ID NO: 688 or SEQ ID NO: 711), MOTIF 14 (SEQ ID NO: 659, SEQ ID NO:683 or SEQ ID NO: 706), MOTIF 2 (SEQ ID NO: 647, SEQ ID NO: 671 or SEQID NO: 694), MOTIF 8 (SEQ ID NO: 653, SEQ ID NO: 677 or SEQ ID NO: 700),MOTIF 15 (SEQ ID NO: 660, SEQ ID NO: 684 or SEQ ID NO: 707), MOTIF 9(SEQ ID NO: 654, SEQ ID NO: 678 or SEQ ID NO: 701), MOTIF 21 (SEQ ID NO:666, SEQ ID NO: 689 or SEQ ID NO: 712), MOTIF 1 (SEQ ID NO: 646, SEQ IDNO: 670 or SEQ ID NO: 693), MOTIF 17 (SEQ ID NO: 662, SEQ ID NO: 686 orSEQ ID NO: 709), MOTIF 6 (SEQ ID NO: 651, SEQ ID NO: 675 or SEQ ID NO:698), MOTIF 12 (SEQ ID NO: 657, SEQ ID NO: 681 or SEQ ID NO: 704), MOTIF4 (SEQ ID NO: 649, SEQ ID NO: 673 or SEQ ID NO: 696), MOTIF 16 (SEQ IDNO: 661, SEQ ID NO: 685 or SEQ ID NO: 708), MOTIF 5 (SEQ ID NO: 650, SEQID NO: 674 or SEQ ID NO: 697), MOTIF 23 (SEQ ID NO: 668, SEQ ID NO: 691or SEQ ID NO: 714), and MOTIF 3 (SEQ ID NO: 648, SEQ ID NO: 672 or SEQID NO: 695).

In some embodiments a PtIP-83 polypeptide comprises, sequentially fromthe N-terminus to the C-terminus, the amino acid sequence MOTIFs: MOTIF19 (SEQ ID NO: 664, SEQ ID NO: 687 or SEQ ID NO: 710), MOTIF 7 (SEQ IDNO: 652, SEQ ID NO: 676 or SEQ ID NO: 699), MOTIF 13 (SEQ ID NO: 658,SEQ ID NO: 682 or SEQ ID NO: 705), MOTIF 20 (SEQ ID NO: 665, SEQ ID NO:688 or SEQ ID NO: 711), MOTIF 10 (SEQ ID NO: 655, SEQ ID NO: 679 or SEQID NO: 702), MOTIF 18 (SEQ ID NO: 663), MOTIF 24 (SEQ ID NO: 669, SEQ IDNO: 692 or SEQ ID NO: 715), MOTIF 14 (SEQ ID NO: 659, SEQ ID NO: 683 orSEQ ID NO: 706), MOTIF 11 (SEQ ID NO: 656, SEQ ID NO: 680 or SEQ ID NO:703), MOTIF 22 (SEQ ID NO: 667, SEQ ID NO: 690 or SEQ ID NO: 713), MOTIF2 (SEQ ID NO: 647, SEQ ID NO: 671 or SEQ ID NO: 694), MOTIF 8 (SEQ IDNO: 653, SEQ ID NO: 677 or SEQ ID NO: 700), MOTIF 15 (SEQ ID NO: 660,SEQ ID NO: 684 or SEQ ID NO: 707), MOTIF 9 (SEQ ID NO: 654, SEQ ID NO:678 or SEQ ID NO: 701), MOTIF 21 (SEQ ID NO: 666, SEQ ID NO: 689 or SEQID NO: 712), MOTIF 1 (SEQ ID NO: 646, SEQ ID NO: 670 or SEQ ID NO: 693),MOTIF 17 (SEQ ID NO: 662, SEQ ID NO: 686 or SEQ ID NO: 709), MOTIF 6(SEQ ID NO: 651, SEQ ID NO: 675 or SEQ ID NO: 698), MOTIF 12 (SEQ ID NO:657, SEQ ID NO: 681 or SEQ ID NO: 704), MOTIF 4 (SEQ ID NO: 649, SEQ IDNO: 673 or SEQ ID NO: 696), MOTIF 16 (SEQ ID NO: 661, SEQ ID NO: 685 orSEQ ID NO: 708), MOTIF 5 (SEQ ID NO: 650, SEQ ID NO: 674 or SEQ ID NO:697), MOTIF 23 (SEQ ID NO: 668, SEQ ID NO: 691 or SEQ ID NO: 714), andMOTIF 3 (SEQ ID NO: 648, SEQ ID NO: 672 or SEQ ID NO: 695).

In some embodiments a PtIP-83 polypeptide comprises, sequentially fromthe N-terminus to the C-terminus, the amino acid sequence MOTIFs: MOTIF19 (SEQ ID NO: 664, SEQ ID NO: 687 or SEQ ID NO: 710), MOTIF 7 (SEQ IDNO: 652, SEQ ID NO: 676 or SEQ ID NO: 699), MOTIF 13 (SEQ ID NO: 658,SEQ ID NO: 682 or SEQ ID NO: 705), MOTIF 20 (SEQ ID NO: 665, SEQ ID NO:688 or SEQ ID NO: 711), MOTIF 14 (SEQ ID NO: 659, SEQ ID NO: 683 or SEQID NO: 706), MOTIF 2 (SEQ ID NO: 647, SEQ ID NO: 671 or SEQ ID NO: 694),MOTIF 8 (SEQ ID NO: 653, SEQ ID NO: 677 or SEQ ID NO: 700), MOTIF 15(SEQ ID NO: 660, SEQ ID NO: 684 or SEQ ID NO: 707), MOTIF 9 (SEQ ID NO:654, SEQ ID NO: 678 or SEQ ID NO: 701), MOTIF 21 (SEQ ID NO: 666, SEQ IDNO: 689 or SEQ ID NO: 712), MOTIF 1 (SEQ ID NO: 646, SEQ ID NO: 670 orSEQ ID NO: 693), MOTIF 17 (SEQ ID NO: 662, SEQ ID NO: 686 or SEQ ID NO:709), MOTIF 6 (SEQ ID NO: 651, SEQ ID NO: 675 or SEQ ID NO: 698), MOTIF12 (SEQ ID NO: 657, SEQ ID NO: 681 or SEQ ID NO: 704), MOTIF 4 (SEQ IDNO: 649, SEQ ID NO: 673 or SEQ ID NO: 696), MOTIF 16 (SEQ ID NO: 661,SEQ ID NO: 685 or SEQ ID NO: 708), MOTIF 5 (SEQ ID NO: 650, SEQ ID NO:674 or SEQ ID NO: 697), MOTIF 23 (SEQ ID NO: 668, SEQ ID NO: 691 or SEQID NO: 714), and MOTIF 3 (SEQ ID NO: 648, SEQ ID NO: 672 or SEQ ID NO:695).

In some embodiments a PtIP-83 polypeptide comprises, sequentially fromthe N-terminus to the C-terminus: a Region A of between about 200 toabout 300 amino acids in length comprising an amino acid sequence MOTIFof: MOTIF 19 (SEQ ID NO: 664, SEQ ID NO: 687 or SEQ ID NO: 710), MOTIF 7(SEQ ID NO: 652, SEQ ID NO: 676 or SEQ ID NO: 699), MOTIF 13 (SEQ ID NO:658, SEQ ID NO: 682 or SEQ ID NO: 705), MOTIF 20 (SEQ ID NO: 665, SEQ IDNO: 688 or SEQ ID NO: 711), MOTIF 10 (SEQ ID NO: 655, SEQ ID NO: 679 orSEQ ID NO: 702), MOTIF 18 (SEQ ID NO: 663), MOTIF 24 (SEQ ID NO: 669,SEQ ID NO: 692 or SEQ ID NO: 715), and/or MOTIF 14 having apredominantly nonconserved secondary structure; a Region B of betweenabout 380 to about 465 amino acids in length comprising an amino acidsequence MOTIF of MOTIF 22 (SEQ ID NO: 667, SEQ ID NO: 690 or SEQ ID NO:713), MOTIF 2 (SEQ ID NO: 647, SEQ ID NO: 671 or SEQ ID NO: 694), MOTIF8 (SEQ ID NO: 653, SEQ ID NO: 677 or SEQ ID NO: 700), MOTIF 15 (SEQ IDNO: 660, SEQ ID NO: 684 or SEQ ID NO: 707), MOTIF 9 (SEQ ID NO: 654, SEQID NO: 678 or SEQ ID NO: 701), MOTIF 21 (SEQ ID NO: 666, SEQ ID NO: 689or SEQ ID NO: 712), MOTIF 1 (SEQ ID NO: 646, SEQ ID NO: 670 or SEQ IDNO: 693), MOTIF 17 (SEQ ID NO: 662, SEQ ID NO: 686 or SEQ ID NO: 709),MOTIF 6 (SEQ ID NO: 651, SEQ ID NO: 675 or SEQ ID NO: 698), and/or MOTIF12 and having a predominately alpha helical structure; and a Region C ofbetween about 150 to about 180 amino acids in length comprising an aminoacid sequence MOTIF of MOTIF 16 (SEQ ID NO: 661, SEQ ID NO: 685 or SEQID NO: 708), MOTIF 5 (SEQ ID NO: 650, SEQ ID NO: 674 or SEQ ID NO: 697),MOTIF 23 (SEQ ID NO: 668, SEQ ID NO: 691 or SEQ ID NO: 714), and/orMOTIF 3 having a consensus secondary structure comprising predominatelybeta strand structure.

In some embodiments a PtIP-83 polypeptide comprises sequentially fromthe N-terminus to the C-terminus: a Region A of between about 200 toabout 300 amino acids in length having predominantly a nonconservedsecondary structure; a Region B of between about 380 to about 465 aminoacids in length having a consensus secondary structure comprising 8 to10 segments of predominately alpha helical structure; and a Region C ofbetween about 150 to about 180 amino acids in length having a consensussecondary structure comprising 6 to 8 segments of predominately betastrand structure. As used herein “predominantly a nonconserved secondarystructure” means that the regions of secondary structure don'tconsistently align within the family of PtIP polypeptides. As usedherein “predominately alpha helical structure” means that secondarystructure prediction may have one or more gap of between 1 to 4 aminoacids of coil and/or beta strand structure intervening in the alphahelix structure. As used herein “predominately beta strand structure”means that secondary structure prediction may have a gap of between 1 to4 amino acids of coil and/or alpha helix structure intervening in thebeta strand structure. In some embodiments the secondary structure isgenerated by the PSIPRED, top ranked secondary structure predictionmethod (Jones D T. (1999) J. Mol. Biol. 292: 195-202).

In some embodiments a PtIP-83 polypeptide comprises sequentially fromthe N-terminus to the C-terminus: a Region A of between about 200 toabout 300 amino acids in length having a predominantly nonconservedsecondary structure; a Region B of between about 380 to about 465 aminoacids in length having a consensus secondary structure comprising ninesegments of predominately alpha helical structure; and a Region C ofbetween about 150 to about 180 amino acids in length having a consensussecondary structure comprising seven segments of predominately betastrand structure.

In some embodiments a PtIP-83 polypeptide comprises sequentially fromthe N-terminus to the C-terminus: a Region A of between about 200 toabout 300 amino acids in length having a flexible consensus secondarystructure, wherein the Region A comprises a conserved beta strand 1(β1a) of between about 4 and about 12 amino acids in length within aboutamino acid residue 30 to about amino acid residue 130 from theN-terminus of the PtIP-83 polypeptide; a Region B of between about 380to about 465 amino acids in length having a consensus secondarystructure comprising nine segments of predominately alpha helicalstructure; and a Region C of between about 150 to about 180 amino acidsin length having a consensus secondary structure comprising sevensegments of predominately beta strand structure. As used herein, theterm “about” when used in the context of the lower/upper limit of thelength of a secondary structural element means the greater of −/+ aninteger of up to −/+20% of the length of the secondary structuralelement or −/+1 amino acid. By means of example, a secondary structureelement of between about 3 amino acids and about 23 amino acids inlength means a secondary structure element of between 2 and 27 aminoacids in length.

In some embodiments a PtIP-83 polypeptide comprises sequentially fromthe N-terminus to the C-terminus: a Region A of between about 200 toabout 300 amino acids in length having a flexible consensus secondarystructure, wherein the Region A comprises a conserved beta strand 1(β1a) of between about 4 and about 12 amino acids in length, a coil ofbetween about 10 and and about 20 amino acids in length and a betastrand 2 (β1b) of between about 4 and about 12 amino acids in length,within about amino acid residue 30 to about amino acid residue 165 fromthe N-terminus of the PtIP-83 polypeptide; a Region B of between about380 to about 465 amino acids in length having a consensus secondarystructure comprising nine segments of predominately alpha helicalstructure; and a Region C of between about 150 to about 180 amino acidsin length having a consensus secondary structure comprising sevensegments of predominately beta strand structure.

In some embodiments a PtIP-83 polypeptide comprises sequentially fromthe N-terminus to the C-terminus: a Region A of between about 200 toabout 300 amino acids in length having a predominantly nonconservedsecondary structure; a Region B of between about 380 to about 465 aminoacids in length having a consensus secondary structure comprisingsequentially: i) an alpha helix-1 of between about 10 and about 26 aminoacids in length; ii) a coil-1 of between about 2 and about 8 amino acidsin length flanked by alpha helix-1 and alpha helix-2; iii) an alphahelix-2 of between about 15 and about 24 amino acids in length; iv) acoil-2 of between about 4 and about 14 amino acids in length flanked byalpha helix-2 and alpha helix-3; v) an alpha helix 3 of between about 15and about 27 amino acids in length; vi) a coil-3 of between about 11 andabout 13 amino acids in length flanked by alpha helix-3 and alphahelix-4; vii) an alpha helix-4 of about 180 amino acids in length; viii)a coil-4 of between about 4 and about 5 amino acids in length flanked byalpha helix-4 and alpha helix-5; ix) an alpha helix-5 of between about50 and about 54 amino acids in length; x) a coil-5 of between about 11and about 17 amino acids in length flanked by alpha helix-5 and alphahelix-6; xi) an alpha helix-6 of between about 15 and about 16 aminoacids in length; xii) a coil-6 of between about 6 and about 9 aminoacids in length flanked by alpha helix-6 and alpha helix-7; xiii) analpha helix-7 of between about 49 and about 55 amino acids in length;xiv) a coil-7 of between about 3 and about 8 amino acids in lengthflanked by alpha helix-7 and alpha helix-8; xv) an alpha helix-8 ofbetween about 33 and about 36 amino acids in length; xvi) a coil-8 ofbetween about 14 and about 16 amino acids in length flanked by alphahelix-8 and alpha helix-9; xvii) an alpha helix-9 of between about 16and about 23 amino acids in length; xviii) a coil-9 of between about 21and about 28 amino acids in length flanked by alpha helix-9 and RegionC; and a Region C of between about 150 to about 180 amino acids inlength having a consensus secondary structure comprising seven segmentsof predominately beta strand structure.

In some embodiments a PtIP-83 polypeptide comprises sequentially fromthe N-terminus to the C-terminus: a Region A of between about 200 toabout 300 amino acids in length having a predominantly nonconservedsecondary structure; a Region B of between about 380 to about 465 aminoacids in length having a consensus secondary structure comprising ninesegments of predominately alpha helical structure; and a Region C ofbetween about 150 to about 180 amino acids in length having a consensussecondary structure comprising sequentially: i) a beta strand-1 (β1) ofbetween about 3 amino acids and about 5 amino acids in length; ii) acoil of between about 13 amino acids and about 17 amino acids in length;iii) a beta strand-2 (β2) of between about 7 amino acids and about 11amino acids in length; iv) a coil of between about 17 amino acids andabout 23 amino acids in length; v) a beta strand-3 (β3) of between about5 amino acids and about 7 amino acids in length; vi) a coil of betweenabout 12 amino acids and about 14 amino acids in length; vii) a betastrand-4 (β4) of between about 5 amino acids and about 6 amino acids inlength; viii) a coil of between about 2 amino acids and about 7 aminoacids in length; ix) a beta strand-5 (β5) of between about 5 amino acidsand about 7 amino acids in length; x) a coil of between about 26 aminoacids and about 28 amino acids in length; xi) a beta strand-6 (β6) ofbetween about 5 amino acids and about 7 amino acids in length; xii) acoil of between about 16 amino acids and about 20 amino acids in length;and xiii) a beta strand-1 (β7) of between about 13 amino acids and about17 amino acids in length.

In some embodiments a PtIP-83 polypeptide comprises sequentially fromthe N-terminus to the C-terminus: a Region A of between about 200 toabout 300 amino acids in length having a predominantly nonconservedsecondary structure; a Region B of between about 380 to about 465 aminoacids in length having a consensus secondary structure comprisingsequentially: i) an alpha helix-1 of between about 10 and about 26 aminoacids in length; ii) a coil-1 of between about 2 and about 8 amino acidsin length flanked by alpha helix-1 and alpha helix-2; iii) an alphahelix-2 of between about 15 and about 24 amino acids in length; iv) acoil-2 of between about 4 and about 14 amino acids in length flanked byalpha helix-2 and alpha helix-3; v) an alpha helix 3 of between about 15and about 27 amino acids in length; vi) a coil-3 of between about 11 andabout 13 amino acids in length flanked by alpha helix-3 and alphahelix-4; vii) an alpha helix-4 of about 24 180 amino acids in length;viii) a coil-4 of between about 4 and about 5 amino acids in lengthflanked by alpha helix-4 and alpha helix-5; ix) an alpha helix-5 ofbetween about 50 and about 54 amino acids in length; x) a coil-5 ofbetween about 11 and about 17 amino acids in length flanked by alphahelix-5 and alpha helix-6; xi) an alpha helix-6 of between about 15 andabout 16 amino acids in length; xii) a coil-6 of between about 6 andabout 9 amino acids in length flanked by alpha helix-6 and alphahelix-7; xiii) an alpha helix-7 of between about 49 and about 55 aminoacids in length; xiv) a coil-7 of between about 3 and about 8 aminoacids in length flanked by alpha helix-7 and alpha helix-8; xv) an alphahelix-8 of between about 33 and about 36 amino acids in length; xvi) acoil-8 of between about 14 and about 16 amino acids in length flanked byalpha helix-8 and alpha helix-9; xvii) an alpha helix-9 of between about16 and about 23 amino acids in length; xviii) a coil-9 of between about21 and about 28 amino acids in length flanked by alpha helix-9 andRegion C; and a Region C of between about 150 to about 180 amino acidsin length having a consensus secondary structure comprisingsequentially: i) a beta strand-1 (β1) of between about 3 amino acids andabout 5 amino acids in length; ii) a coil of between about 13 aminoacids and about 17 amino acids in length; iii) a beta strand-2 (β2) ofbetween about 7 amino acids and about 11 amino acids in length; iv) acoil of between about 17 amino acids and about 23 amino acids in length;v) a beta strand-3 (β3) of between about 5 amino acids and about 7 aminoacids in length; vi) a coil of between about 12 amino acids and about 14amino acids in length; vii) a beta strand-4 (β4) of between about 5amino acids and about 6 amino acids in length; viii) a coil of betweenabout 2 amino acids and about 7 amino acids in length; ix) a betastrand-5 (β5) of between about 5 amino acids and about 7 amino acids inlength; x) a coil of between about 26 amino acids and about 28 aminoacids in length; xi) a beta strand-6 (β6) of between about 5 amino acidsand about 7 amino acids in length; xii) a coil of between about 16 aminoacids and about 20 amino acids in length; and xiii) a beta strand-1 (β7)of between about 13 amino acids and about 17 amino acids in length.

In some embodiments a PtIP-83 polypeptide comprises sequentially fromthe N-terminus to the C-terminus: a Region A of between about 200 toabout 300 amino acids in length having a flexible consensus secondarystructure, wherein the Region A comprises a conserved beta strand 1(β1a) of between about 4 and about 12 amino acids in length within aboutamino acid residue 30 to about amino acid residue 130 from theN-terminus of the PtIP-83 polypeptide; a Region B of between about 380to about 465 amino acids in length having a consensus secondarystructure comprising sequentially: i) an alpha helix-1 of between about10 and about 26 amino acids in length; ii) a coil-1 of between about 2and about 8 amino acids in length flanked by alpha helix-1 and alphahelix-2; iii) an alpha helix-2 of between about 15 and about 24 aminoacids in length; iv) a coil-2 of between about 4 and about 14 aminoacids in length flanked by alpha helix-2 and alpha helix-3; v) an alphahelix 3 of between about 15 and about 27 amino acids in length; vi) acoil-3 of between about 11 and about 13 amino acids in length flanked byalpha helix-3 and alpha helix-4; vii) an alpha helix-4 of about 24 180amino acids in length; viii) a coil-4 of between about 4 and about 5amino acids in length flanked by alpha helix-4 and alpha helix-5; ix) analpha helix-5 of between about 50 and about 54 amino acids in length; x)a coil-5 of between about 11 and about 17 amino acids in length flankedby alpha helix-5 and alpha helix-6; xi) an alpha helix-6 of betweenabout 15 and about 16 amino acids in length; xii) a coil-6 of betweenabout 6 and about 9 amino acids in length flanked by alpha helix-6 andalpha helix-7; xiii) an alpha helix-7 of between about 49 and about 55amino acids in length; xiv) a coil-7 of between about 3 and about 8amino acids in length flanked by alpha helix-7 and alpha helix-8; xv) analpha helix-8 of between about 33 and about 36 amino acids in length;xvi) a coil-8 of between about 14 and about 16 amino acids in lengthflanked by alpha helix-8 and alpha helix-9; xvii) an alpha helix-9 ofbetween about 16 and about 23 amino acids in length; xviii) a coil-9 ofbetween about 21 and about 28 amino acids in length flanked by alphahelix-9 and Region C; and a Region C of between about 150 to about 180amino acids in length having a consensus secondary structure comprisingsequentially: i) a beta strand-1 (β1) of between about 3 amino acids andabout 5 amino acids in length; ii) a coil of between about 13 aminoacids and about 17 amino acids in length; iii) a beta strand-2 (β2) ofbetween about 7 amino acids and about 11 amino acids in length; iv) acoil of between about 17 amino acids and about 23 amino acids in length;v) a beta strand-3 (β3) of between about 5 amino acids and about 7 aminoacids in length; vi) a coil of between about 12 amino acids and about 14amino acids in length; vii) a beta strand-4 (β4) of between about 5amino acids and about 6 amino acids in length; viii) a coil of betweenabout 2 amino acids and about 7 amino acids in length; ix) a betastrand-5 (β5) of between about 5 amino acids and about 7 amino acids inlength; x) a coil of between about 26 amino acids and about 28 aminoacids in length; xi) a beta strand-6 (β6) of between about 5 amino acidsand about 7 amino acids in length; xii) a coil of between about 16 aminoacids and about 20 amino acids in length; and xiii) a beta strand-1 (β7)of between about 13 amino acids and about 17 amino acids in length.

In some embodiments a PtIP-83 polypeptide has a calculated molecularweight of between about 70 kD and about 120 kD, between about 75 kD andabout 110 kD, and between about 80 kD and about 105 kD, and betweenabout 85 kD and about 105 kD.

In some embodiments the PtIP-83 polypeptide has a modified physicalproperty. As used herein, the term “physical property” refers to anyparameter suitable for describing the physical-chemical characteristicsof a protein. As used herein, “physical property of interest” and“property of interest” are used interchangeably to refer to physicalproperties of proteins that are being investigated and/or modified.Examples of physical properties include, but are not limited to netsurface charge and charge distribution on the protein surface, nethydrophobicity and hydrophobic residue distribution on the proteinsurface, surface charge density, surface hydrophobicity density, totalcount of surface ionizable groups, surface tension, protein size and itsdistribution in solution, melting temperature, heat capacity, and secondvirial coefficient. Examples of physical properties also include, butare not limited to solubility, folding, stability, and digestibility. Insome embodiments the PtIP-83 polypeptide has increased digestibility ofproteolytic fragments in an insect gut. Models for digestion bysimulated simulated gastric fluids are known to one skilled in the art(Fuchs, R. L. and J. D. Astwood. Food Technology 50: 83-88, 1996;Astwood, J. D., et al Nature Biotechnology 14: 1269-1273, 1996; Fu T Jet al J. Agric Food Chem. 50: 7154-7160, 2002).

In some embodiments variants include polypeptides that differ in aminoacid sequence due to mutagenesis. Variant proteins encompassed by thedisclosure are biologically active, that is they continue to possess thedesired biological activity (i.e. pesticidal activity) of the nativeprotein. In some embodiment the variant will have at least about 10%, atleast about 30%, at least about 50%, at least about 70%, at least about80% or more of the insecticidal activity of the native protein. In someembodiments, the variants may have improved activity over the nativeprotein.

Bacterial genes quite often possess multiple methionine initiationcodons in proximity to the start of the open reading frame. Often,translation initiation at one or more of these start codons will lead togeneration of a functional protein. These start codons can include ATGcodons. However, bacteria such as Bacillus sp. also recognize the codonGTG as a start codon, and proteins that initiate translation at GTGcodons contain a methionine at the first amino acid. On rare occasions,translation in bacterial systems can initiate at a TTG codon, though inthis event the TTG encodes a methionine. Furthermore, it is not oftendetermined a priori which of these codons are used naturally in thebacterium. Thus, it is understood that use of one of the alternatemethionine codons may also lead to generation of pesticidal proteins.These pesticidal proteins are encompassed in the present disclosure andmay be used in the methods of the present disclosure. It will beunderstood that, when expressed in plants, it will be necessary to alterthe alternate start codon to ATG for proper translation.

In another aspect the PtIP-83 polypeptide may be expressed as aprecursor protein with an intervening sequence that catalyzesmulti-step, post translational protein splicing. Protein splicinginvolves the excision of an intervening sequence from a polypeptide withthe concomitant joining of the flanking sequences to yield a newpolypeptide (Chong, et al., (1996) J. Biol. Chem., 271:22159-22168).This intervening sequence or protein splicing element, referred to asinteins, which catalyze their own excision through three coordinatedreactions at the N-terminal and C-terminal splice junctions: an acylrearrangement of the N-terminal cysteine or serine; a transesterficationreaction between the two termini to form a branched ester or thioesterintermediate and peptide bond cleavage coupled to cyclization of theintein C-terminal asparagine to free the intein (Evans, et al., (2000)J. Biol. Chem., 275:9091-9094. The elucidation of the mechanism ofprotein splicing has led to a number of intein-based applications (Comb,et al., U.S. Pat. No. 5,496,714; Comb, et al., U.S. Pat. No. 5,834,247;Camarero and Muir, (1999) J. Amer. Chem. Soc. 121:5597-5598; Chong, etal., (1997) Gene 192:271-281, Chong, et al., (1998) Nucleic Acids Res.26:5109-5115; Chong, et al., (1998) J. Biol. Chem. 273:10567-10577;Cotton, et al., (1999) J. Am. Chem. Soc. 121:1100-1101; Evans, et al.,(1999) J. Biol. Chem. 274:18359-18363; Evans, et al., (1999) J. Biol.Chem. 274:3923-3926; Evans, et al., (1998) Protein Sci. 7:2256-2264;Evans, et al., (2000) J. Biol. Chem. 275:9091-9094; Iwai and Pluckthun,(1999) FEBS Lett. 459:166-172; Mathys, et al., (1999) Gene 231:1-13;Mills, et al., (1998) Proc. Natl. Acad. Sci. USA 95:3543-3548; Muir, etal., (1998) Proc. Natl. Acad. Sci. USA 95:6705-6710; Otomo, et al.,(1999) Biochemistry 38:16040-16044; Otomo, et al., (1999) J. Biolmol.NMR 14:105-114; Scott, et al., (1999) Proc. Natl. Acad. Sci. USA96:13638-13643; Severinov and Muir, (1998) J. Biol. Chem.273:16205-16209; Shingledecker, et al., (1998) Gene 207:187-195;Southworth, et al., (1998) EMBO J. 17:918-926; Southworth, et al.,(1999) Biotechniques 27:110-120; Wood, et al., (1999) Nat. Biotechnol.17:889-892; Wu, et al., (1998a) Proc. Natl. Acad. Sci. USA 95:9226-9231;Wu, et al., (1998b) Biochim Biophys Acta 1387:422-432; Xu, et al.,(1999) Proc. Natl. Acad. Sci. USA 96:388-393; Yamazaki, et al., (1998)J. Am. Chem. Soc., 120:5591-5592). For the application of inteins inplant transgenes, see, Yang, et al., (Transgene Res 15:583-593 (2006))and Evans, et al., (Annu. Rev. Plant Biol. 56:375-392 (2005)).

In another aspect the PtIP-83 polypeptide may be encoded by two separategenes where the intein of the precursor protein comes from the twogenes, referred to as a split-intein, and the two portions of theprecursor are joined by a peptide bond formation. This peptide bondformation is accomplished by intein-mediated trans-splicing. For thispurpose, a first and a second expression cassette comprising the twoseparate genes further code for inteins capable of mediating proteintrans-splicing. By trans-splicing, the proteins and polypeptides encodedby the first and second fragments may be linked by peptide bondformation. Trans-splicing inteins may be selected from the nucleolar andorganellar genomes of different organisms including eukaryotes,archaebacteria and eubacteria. Inteins that may be used for are listedat neb.com/neb/inteins.html, which can be accessed on the world-wide webusing the “www” prefix). The nucleotide sequence coding for an inteinmay be split into a 5′ and a 3′ part that code for the 5′ and the 3′part of the intein, respectively. Sequence portions not necessary forintein splicing (e.g. homing endonuclease domain) may be deleted. Theintein coding sequence is split such that the 5′ and the 3′ parts arecapable of trans-splicing. For selecting a suitable splitting site ofthe intein coding sequence, the considerations published by Southworth,et al., (1998) EMBO J. 17:918-926 may be followed. In constructing thefirst and the second expression cassette, the 5′ intein coding sequenceis linked to the 3′ end of the first fragment coding for the N-terminalpart of the PtIP-83 polypeptide and the 3′ intein coding sequence islinked to the 5′ end of the second fragment coding for the C-terminalpart of the PtIP-83 polypeptide.

In general, the trans-splicing partners can be designed using any splitintein, including any naturally-occurring or artificially-split splitintein. Several naturally-occurring split inteins are known, forexample: the split intein of the DnaE gene of Synechocystis sp. PCC6803(see, Wu, et al., (1998) Proc Natl Acad Sci USA. 95(16):9226-31 andEvans, et al., (2000) J Biol Chem. 275(13):9091-4 and of the DnaE genefrom Nostoc punctiforme (see, Iwai, et al., (2006) FEBS Lett.580(7):1853-8). Non-split inteins have been artificially split in thelaboratory to create new split inteins, for example: the artificiallysplit Ssp DnaB intein (see, Wu, et al., (1998) Biochim Biophys Acta.1387:422-32) and split Sce VMA intein (see, Brenzel, et al., (2006)Biochemistry. 45(6):1571-8) and an artificially split fungal mini-intein(see, Elleuche, et al., (2007) Biochem Biophys Res Commun.355(3):830-4). There are also intein databases available that catalogueknown inteins (see for example the online-database available at:bioinformatics.weizmann.ac.il/^(˜)pietro/inteins/Inteinstable.html,which can be accessed on the world-wide web using the “www” prefix).

Naturally-occurring non-split inteins may have endonuclease or otherenzymatic activities that can typically be removed when designing anartificially-split split intein. Such mini-inteins or minimized splitinteins are well known in the art and are typically less than 200 aminoacid residues long (see, Wu, et al., (1998) Biochim Biophys Acta.1387:422-32). Suitable split inteins may have other purificationenabling polypeptide elements added to their structure, provided thatsuch elements do not inhibit the splicing of the split intein or areadded in a manner that allows them to be removed prior to splicing.Protein splicing has been reported using proteins that comprisebacterial intein-like (BIL) domains (see, Amitai, et al., (2003) MolMicrobiol. 47:61-73) and hedgehog (Hog) auto-processing domains (thelatter is combined with inteins when referred to as the Hog/inteinsuperfamily or HINT family (see, Dassa, et al., (2004) J Biol Chem.279:32001-7) and domains such as these may also be used to prepareartificially-split inteins. In particular, non-splicing members of suchfamilies may be modified by molecular biology methodologies to introduceor restore splicing activity in such related species. Recent studiesdemonstrate that splicing can be observed when a N-terminal split inteincomponent is allowed to react with a C-terminal split intein componentnot found in nature to be its “partner”; for example, splicing has beenobserved utilizing partners that have as little as 30 to 50% homologywith the “natural” splicing partner (see, Dassa, et al., (2007)Biochemistry. 46(1):322-30). Other such mixtures of disparate splitintein partners have been shown to be unreactive one with another (see,Brenzel, et al., (2006) Biochemistry. 45(6):1571-8). However, it iswithin the ability of a person skilled in the relevant art to determinewhether a particular pair of polypeptides is able to associate with eachother to provide a functional intein, using routine methods and withoutthe exercise of inventive skill.

In another aspect the PtIP-83 polypeptide is a circular permutedvariant. In certain embodiments the PtIP-83 polypeptide is a circularpermuted variant of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 3, SEQID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ IDNO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761,SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ IDNO: 766, SEQ ID NO: 767, SEQ ID NO: 768 or SEQ ID NO: 769.

The development of recombinant DNA methods has made it possible to studythe effects of sequence transposition on protein folding, structure andfunction. The approach used in creating new sequences resembles that ofnaturally occurring pairs of proteins that are related by linearreorganization of their amino acid sequences (Cunningham, et al., (1979)Proc. Natl. Acad. Sci. U.S.A. 76:3218-3222; Teather and Erfle, (1990) J.Bacteriol. 172:3837-3841; Schimming, et al., (1992) Eur. J. Biochem.204:13-19; Yamiuchi and Minamikawa, (1991) FEBS Lett. 260:127-130;MacGregor, et al., (1996) FEBS Lett. 378:263-266). The first in vitroapplication of this type of rearrangement to proteins was described byGoldenberg and Creighton (J. Mol. Biol. 165:407-413, 1983). In creatinga circular permuted variant a new N-terminus is selected at an internalsite (breakpoint) of the original sequence, the new sequence having thesame order of amino acids as the original from the breakpoint until itreaches an amino acid that is at or near the original C-terminus. Atthis point the new sequence is joined, either directly or through anadditional portion of sequence (linker), to an amino acid that is at ornear the original N-terminus and the new sequence continues with thesame sequence as the original until it reaches a point that is at ornear the amino acid that was N-terminal to the breakpoint site of theoriginal sequence, this residue forming the new C-terminus of the chain.The length of the amino acid sequence of the linker can be selectedempirically or with guidance from structural information or by using acombination of the two approaches. When no structural information isavailable, a small series of linkers can be prepared for testing using adesign whose length is varied in order to span a range from 0 to 50 Åand whose sequence is chosen in order to be consistent with surfaceexposure (hydrophilicity, Hopp and Woods, (1983) Mol. Immunol.20:483-489; Kyte and Doolittle, (1982) J. Mol. Biol. 157:105-132;solvent exposed surface area, Lee and Richards, (1971) J. Mol. Biol.55:379-400) and the ability to adopt the necessary conformation withoutderanging the configuration of the pesticidal polypeptide(conformationally flexible; Karplus and Schulz, (1985)Naturwissenschaften 72:212-213). Assuming an average of translation of2.0 to 3.8 Å per residue, this would mean the length to test would bebetween 0 to 30 residues, with 0 to 15 residues being the preferredrange. Exemplary of such an empirical series would be to constructlinkers using a cassette sequence such as Gly-Gly-Gly-Ser repeated ntimes, where n is 1, 2, 3 or 4. Those skilled in the art will recognizethat there are many such sequences that vary in length or compositionthat can serve as linkers with the primary consideration being that theybe neither excessively long nor short (cf., Sandhu, (1992) Critical Rev.Biotech. 12:437-462); if they are too long, entropy effects will likelydestabilize the three-dimensional fold, and may also make foldingkinetically impractical, and if they are too short, they will likelydestabilize the molecule because of torsional or steric strain. Thoseskilled in the analysis of protein structural information will recognizethat using the distance between the chain ends, defined as the distancebetween the c-alpha carbons, can be used to define the length of thesequence to be used or at least to limit the number of possibilitiesthat must be tested in an empirical selection of linkers. They will alsorecognize that it is sometimes the case that the positions of the endsof the polypeptide chain are ill-defined in structural models derivedfrom x-ray diffraction or nuclear magnetic resonance spectroscopy data,and that when true, this situation will therefore need to be taken intoaccount in order to properly estimate the length of the linker required.From those residues whose positions are well defined are selected tworesidues that are close in sequence to the chain ends, and the distancebetween their c-alpha carbons is used to calculate an approximate lengthfor a linker between them. Using the calculated length as a guide,linkers with a range of number of residues (calculated using 2 to 3.8 Åper residue) are then selected. These linkers may be composed of theoriginal sequence, shortened or lengthened as necessary, and whenlengthened the additional residues may be chosen to be flexible andhydrophilic as described above; or optionally the original sequence maybe substituted for using a series of linkers, one example being theGly-Gly-Gly-Ser cassette approach mentioned above; or optionally acombination of the original sequence and new sequence having theappropriate total length may be used. Sequences of pesticidalpolypeptides capable of folding to biologically active states can beprepared by appropriate selection of the beginning (amino terminus) andending (carboxyl terminus) positions from within the originalpolypeptide chain while using the linker sequence as described above.Amino and carboxyl termini are selected from within a common stretch ofsequence, referred to as a breakpoint region, using the guidelinesdescribed below. A novel amino acid sequence is thus generated byselecting amino and carboxyl termini from within the same breakpointregion. In many cases the selection of the new termini will be such thatthe original position of the carboxyl terminus immediately preceded thatof the amino terminus. However, those skilled in the art will recognizethat selections of termini anywhere within the region may function, andthat these will effectively lead to either deletions or additions to theamino or carboxyl portions of the new sequence. It is a central tenet ofmolecular biology that the primary amino acid sequence of a proteindictates folding to the three-dimensional structure necessary forexpression of its biological function. Methods are known to thoseskilled in the art to obtain and interpret three-dimensional structuralinformation using x-ray diffraction of single protein Crystals ornuclear magnetic resonance spectroscopy of protein solutions. Examplesof structural information that are relevant to the identification ofbreakpoint regions include the location and type of protein secondarystructure (alpha and 3-10 helices, parallel and anti-parallel betasheets, chain reversals and turns, and loops; Kabsch and Sander, (1983)Biopolymers 22:2577-2637; the degree of solvent exposure of amino acidresidues, the extent and type of interactions of residues with oneanother (Chothia, (1984) Ann. Rev. Biochem. 53:537-572) and the staticand dynamic distribution of conformations along the polypeptide chain(Alber and Mathews, (1987) Methods Enzymol. 154:511-533). In some casesadditional information is known about solvent exposure of residues; oneexample is a site of post-translational attachment of carbohydrate whichis necessarily on the surface of the protein. When experimentalstructural information is not available or is not feasible to obtain,methods are also available to analyze the primary amino acid sequence inorder to make predictions of protein tertiary and secondary structure,solvent accessibility and the occurrence of turns and loops. Biochemicalmethods are also sometimes applicable for empirically determiningsurface exposure when direct structural methods are not feasible; forexample, using the identification of sites of chain scission followinglimited proteolysis in order to infer surface exposure (Gentile andSalvatore, (1993) Eur. J. Biochem. 218:603-621). Thus using either theexperimentally derived structural information or predictive methods(e.g., Srinivisan and Rose, (1995) Proteins: Struct., Funct. & Genetics22:81-99) the parental amino acid sequence is inspected to classifyregions according to whether or not they are integral to the maintenanceof secondary and tertiary structure. The occurrence of sequences withinregions that are known to be involved in periodic secondary structure(alpha and 3-10 helices, parallel and anti-parallel beta sheets) areregions that should be avoided. Similarly, regions of amino acidsequence that are observed or PtIP- to have a low degree of solventexposure are more likely to be part of the so-called hydrophobic core ofthe protein and should also be avoided for selection of amino andcarboxyl termini. In contrast, those regions that are known or PtIP- tobe in surface turns or loops, and especially those regions that areknown not to be required for biological activity, are the preferredsites for location of the extremes of the polypeptide chain. Continuousstretches of amino acid sequence that are preferred based on the abovecriteria are referred to as a breakpoint region. Polynucleotidesencoding circular permuted PtIP-83 polypeptides with newN-terminus/C-terminus which contain a linker region separating theoriginal C-terminus and N-terminus can be made essentially following themethod described in Mullins, et al., (1994) J. Am. Chem. Soc.116:5529-5533. Multiple steps of polymerase chain reaction (PCR)amplifications are used to rearrange the DNA sequence encoding theprimary amino acid sequence of the protein. Polynucleotides encodingcircular permuted PtIP-83 polypeptides with new N-terminus/C-terminuswhich contain a linker region separating the original C-terminus andN-terminus can be made based on the tandem-duplication method describedin Horlick, et al., (1992) Protein Eng. 5:427-431. Polymerase chainreaction (PCR) amplification of the new N-terminus/C-terminus genes isperformed using a tandemly duplicated template DNA.

In another aspect fusion proteins are provided that include within itsamino acid sequence an amino acid sequence comprising a PtIP-83polypeptide including but not limited to the polypeptide of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ IDNO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755,SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ IDNO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID NO: 764,SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768, SEQ IDNO: 769, and active fragments thereof.

Methods for design and construction of fusion proteins (andpolynucleotides encoding same) are known to those of skill in the art.Polynucleotides encoding a PtIP-83 polypeptide may be fused to signalsequences which will direct the localization of the PtIP-83 polypeptideto particular compartments of a prokaryotic or eukaryotic cell and/ordirect the secretion of the PtIP-83 polypeptide of the embodiments froma prokaryotic or eukaryotic cell. For example, in E. coli, one may wishto direct the expression of the protein to the periplasmic space.Examples of signal sequences or proteins (or fragments thereof) to whichthe PtIP-83 polypeptide may be fused in order to direct the expressionof the polypeptide to the periplasmic space of bacteria include, but arenot limited to, the pelB signal sequence, the maltose binding protein(MBP) signal sequence, MBP, the ompA signal sequence, the signalsequence of the periplasmic E. coli heat-labile enterotoxin B-subunitand the signal sequence of alkaline phosphatase. Several vectors arecommercially available for the construction of fusion proteins whichwill direct the localization of a protein, such as the pMAL series ofvectors (particularly the pMAL-p series) available from New EnglandBiolabs. In a specific embodiment, the PtIP-83 polypeptide may be fusedto the pelB pectate lyase signal sequence to increase the efficiency ofexpression and purification of such polypeptides in Gram-negativebacteria (see, U.S. Pat. Nos. 5,576,195 and 5,846,818). Plant plastidtransit peptide/polypeptide fusions are well known in the art (see, U.S.Pat. No. 7,193,133). Apoplast transit peptides such as rice or barleyalpha-amylase secretion signal are also well known in the art. Theplastid transit peptide is generally fused N-terminal to the polypeptideto be targeted (e.g., the fusion partner). In one embodiment, the fusionprotein consists essentially of the plastid transit peptide and thePtIP-83 polypeptide to be targeted. In another embodiment, the fusionprotein comprises the plastid transit peptide and the polypeptide to betargeted. In such embodiments, the plastid transit peptide is preferablyat the N-terminus of the fusion protein. However, additional amino acidresidues may be N-terminal to the plastid transit peptide providing thatthe fusion protein is at least partially targeted to a plastid. In aspecific embodiment, the plastid transit peptide is in the N-terminalhalf, N-terminal third or N-terminal quarter of the fusion protein. Mostor all of the plastid transit peptide is generally cleaved from thefusion protein upon insertion into the plastid. The position of cleavagemay vary slightly between plant species, at different plantdevelopmental stages, as a result of specific intercellular conditionsor the particular combination of transit peptide/fusion partner used. Inone embodiment, the plastid transit peptide cleavage is homogenous suchthat the cleavage site is identical in a population of fusion proteins.In another embodiment, the plastid transit peptide is not homogenous,such that the cleavage site varies by 1-10 amino acids in a populationof fusion proteins. The plastid transit peptide can be recombinantlyfused to a second protein in one of several ways. For example, arestriction endonuclease recognition site can be introduced into thenucleotide sequence of the transit peptide at a position correspondingto its C-terminal end and the same or a compatible site can beengineered into the nucleotide sequence of the protein to be targeted atits N-terminal end. Care must be taken in designing these sites toensure that the coding sequences of the transit peptide and the secondprotein are kept “in frame” to allow the synthesis of the desired fusionprotein. In some cases, it may be preferable to remove the initiatormethionine codon of the second protein when the new restriction site isintroduced. The introduction of restriction endonuclease recognitionsites on both parent molecules and their subsequent joining throughrecombinant DNA techniques may result in the addition of one or moreextra amino acids between the transit peptide and the second protein.This generally does not affect targeting activity as long as the transitpeptide cleavage site remains accessible and the function of the secondprotein is not altered by the addition of these extra amino acids at itsN-terminus. Alternatively, one skilled in the art can create a precisecleavage site between the transit peptide and the second protein (withor without its initiator methionine) using gene synthesis (Stemmer, etal., (1995) Gene 164:49-53) or similar methods. In addition, the transitpeptide fusion can intentionally include amino acids downstream of thecleavage site. The amino acids at the N-terminus of the mature proteincan affect the ability of the transit peptide to target proteins toplastids and/or the efficiency of cleavage following protein import.This may be dependent on the protein to be targeted. See, e.g., Comai,et al., (1988) J. Biol. Chem. 263(29):15104-9.

In some embodiments fusion proteins are provide comprising a PtIP-83polypeptide and an insecticidal polypeptide joined by an amino acidlinker. In some embodiments fusion proteins are provided represented bya formula selected from the group consisting of:R¹-L-R²,R²-L-R¹,R¹-R² or R²-R¹wherein R¹ is a PtIP-83 polypeptide, R² is a protein of interest. The R¹polypeptide is fused either directly or through a linker (L) segment tothe R² polypeptide. The term “directly” defines fusions in which thepolypeptides are joined without a peptide linker. Thus “L” represents achemical bound or polypeptide segment to which both R¹ and R² are fusedin frame, most commonly L is a linear peptide to which R¹ and R² arebound by amide bonds linking the carboxy terminus of R¹ to the aminoterminus of L and carboxy terminus of L to the amino terminus of R². By“fused in frame” is meant that there is no translation termination ordisruption between the reading frames of R¹ and R². The linking group(L) is generally a polypeptide of between 1 and 500 amino acids inlength. The linkers joining the two molecules are preferably designed to(1) allow the two molecules to fold and act independently of each other,(2) not have a propensity for developing an ordered secondary structurewhich could interfere with the functional domains of the two proteins,(3) have minimal hydrophobic or charged characteristic which couldinteract with the functional protein domains and (4) provide stericseparation of R¹ and R² such that R¹ and R² could interactsimultaneously with their corresponding receptors on a single cell.Typically surface amino acids in flexible protein regions include Gly,Asn and Ser. Virtually any permutation of amino acid sequencescontaining Gly, Asn and Ser would be expected to satisfy the abovecriteria for a linker sequence. Other neutral amino acids, such as Thrand Ala, may also be used in the linker sequence. Additional amino acidsmay also be included in the linkers due to the addition of uniquerestriction sites in the linker sequence to facilitate construction ofthe fusions.

In some embodiments the linkers comprise sequences selected from thegroup of formulas: (Gly₃Ser)_(n), (Gly₄Ser)_(n), (Gly₅Ser)_(n),(Gly_(n)Ser)_(n) or (AlaGlySer)_(n) where n is an integer. One exampleof a highly-flexible linker is the (GlySer)-rich spacer region presentwithin the pill protein of the filamentous bacteriophages, e.g.bacteriophages M13 or fd (Schaller, et al., 1975). This region providesa long, flexible spacer region between two domains of the pill surfaceprotein. Also included are linkers in which an endopeptidase recognitionsequence is included. Such a cleavage site may be valuable to separatethe individual components of the fusion to determine if they areproperly folded and active in vitro. Examples of various endopeptidasesinclude, but are not limited to, Plasmin, Enterokinase, Kallikerin,Urokinase, Tissue Plasminogen activator, clostripain, Chymosin,Collagenase, Russell's Viper Venom Protease, Postproline cleavageenzyme, V8 protease, Thrombin and factor Xa. In some embodiments thelinker comprises the amino acids EEKKN (SEQ ID NO: 37) from themulti-gene expression vehicle (MGEV), which is cleaved by vacuolarproteases as disclosed in US Patent Application Publication Number US2007/0277263. In other embodiments, peptide linker segments from thehinge region of heavy chain immunoglobulins IgG, IgA, IgM, IgD or IgEprovide an angular relationship between the attached polypeptides.Especially useful are those hinge regions where the cysteines arereplaced with serines. Linkers of the present disclosure includesequences derived from murine IgG gamma 2b hinge region in which thecysteines have been changed to serines. The fusion proteins are notlimited by the form, size or number of linker sequences employed and theonly requirement of the linker is that functionally it does notinterfere adversely with the folding and function of the individualmolecules of the fusion.

In another aspect chimeric PtIP-83 polypeptides are provided that arecreated through joining two or more portions of PtIP-83 genes, whichoriginally encoded separate PtIP-83 proteins to create a chimeric gene.The translation of the chimeric gene results in a single chimericPtIP-83 polypeptide with regions, motifs or domains derived from each ofthe original polypeptides. In certain embodiments the chimeric proteincomprises portions, motifs or domains of PtIP-83 polypeptides of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO:755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID NO:764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768 orSEQ ID NO: 769, in any combination.

It is recognized that DNA sequences may be altered by various methods,and that these alterations may result in DNA sequences encoding proteinswith amino acid sequences different than that encoded by the wild-type(or native) pesticidal protein. In some embodiments a PtIP-83polypeptide may be altered in various ways including amino acidsubstitutions, deletions, truncations and insertions of one or moreamino acids, including up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120,125, 130, 135, 140, 145 or more amino acid substitutions, deletionsand/or insertions or combinations thereof compared to any one of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO:755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID NO:764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768 orSEQ ID NO: 769.

In some embodiments a PtIP-83 polypeptide variant comprises one or moreamino acid substitution, of Table 13, Table 14, Table 15, Table 16,Table 17, Table 18, Table 20, Table 21, Table 23, Table 24 orcombinations thereof, compared to the native amino acid of PtIP-83Aa(SEQ ID NO: 1) at the corresponding residue.

In some embodiments a PtIP-83 polypeptide variant is selected from butnot limited to any one of SEQ ID NO: 236-299, SEQ ID NO: 334-367, SEQ IDNO: 398-427, SEQ ID NO: 518-607, and SEQ ID NO: 728-737.

In some embodiments a PtIP-83 polypeptide comprises a deletion of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids from theN-terminus of the PtIP-83 polypeptide relative to the amino acidposition of any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO:758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO:767, SEQ ID NO: 768 or SEQ ID NO: 769.

In some embodiments a PtIP-83 polypeptide comprises a deletion of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids from theC-terminus of the PtIP-83 polypeptide relative to the amino acidposition of any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO:758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO:767, SEQ ID NO: 768 or SEQ ID NO: 769.

Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of a PtIP-83 polypeptide can beprepared by mutations in the DNA. This may also be accomplished by oneof several forms of mutagenesis and/or in directed evolution. In someaspects, the changes encoded in the amino acid sequence will notsubstantially affect the function of the protein. Such variants willpossess the desired pesticidal activity. However, it is understood thatthe ability of a PtIP-83 polypeptide to confer pesticidal activity maybe improved by the use of such techniques upon the compositions of thisdisclosure.

For example, conservative amino acid substitutions may be made at one ormore, PtIP-, nonessential amino acid residues. A “nonessential” aminoacid residue is a residue that can be altered from the wild-typesequence of a PtIP-83 without altering the biological activity. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include: amino acids with basicside chains (e.g., lysine, arginine, histidine); acidic side chains(e.g., aspartic acid, glutamic acid); polar, negatively charged residuesand their amides (e.g., aspartic acid, asparagine, glutamic, acid,glutamine; uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine); small aliphatic,nonpolar or slightly polar residues (e.g., Alanine, serine, threonine,proline, glycine); nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); largealiphatic, nonpolar residues (e.g., methionine, leucine, isoleucine,valine, cystine); beta-branched side chains (e.g., threonine, valine,isoleucine); aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine); large aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan).

Amino acid substitutions may be made in nonconserved regions that retainfunction. In general, such substitutions would not be made for conservedamino acid residues or for amino acid residues residing within aconserved motif, where such residues are essential for protein activity.Examples of residues that are conserved and that may be essential forprotein activity include, for example, residues that are identicalbetween all proteins contained in an alignment of similar or relatedtoxins to the sequences of the embodiments (e.g., residues that areidentical in an alignment of homologous proteins). Examples of residuesthat are conserved but that may allow conservative amino acidsubstitutions and still retain activity include, for example, residuesthat have only conservative substitutions between all proteins containedin an alignment of similar or related toxins to the sequences of theembodiments (e.g., residues that have only conservative substitutionsbetween all proteins contained in the alignment homologous proteins).However, one of skill in the art would understand that functionalvariants may have minor conserved or nonconserved alterations in theconserved residues. Guidance as to appropriate amino acid substitutionsthat do not affect biological activity of the protein of interest may befound in the model of Dayhoff, et al., (1978) Atlas of Protein Sequenceand Structure (Natl. Biomed. Res. Found., Washington, D.C.), hereinincorporated by reference. In making such changes, the hydropathic indexof amino acids may be considered. The importance of the hydropathicamino acid index in conferring interactive biologic function on aprotein is generally understood in the art (Kyte and Doolittle, (1982) JMol Biol. 157(1):105-32). It is accepted that the relative hydropathiccharacter of the amino acid contributes to the secondary structure ofthe resultant protein, which in turn defines the interaction of theprotein with other molecules, for example, enzymes, substrates,receptors, DNA, antibodies, antigens, and the like.

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. Each amino acid has beenassigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte and Doolittle, ibid). These are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9) and arginine(−4.5). In making such changes, the substitution of amino acids whosehydropathic indices are within +2 is preferred, those which are within+1 are particularly preferred, and those within +0.5 are even moreparticularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, states that the greatest local average hydrophilicity ofa protein, as governed by the hydrophilicity of its adjacent aminoacids, correlates with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0.+0.1); glutamate (+3.0.+0.1); serine(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine(−0.4); proline (−0.5.+0.1); alanine (−0.5); histidine (−0.5); cysteine(−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine(−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

Alternatively, alterations may be made to the protein sequence of manyproteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include insertions, deletions oralterations introduced by modern molecular methods, such as PCR,including PCR amplifications that alter or extend the protein codingsequence by virtue of inclusion of amino acid encoding sequences in theoligonucleotides utilized in the PCR amplification. Alternatively, theprotein sequences added can include entire protein-coding sequences,such as those used commonly in the art to generate protein fusions. Suchfusion proteins are often used to (1) increase expression of a proteinof interest (2) introduce a binding domain, enzymatic activity orepitope to facilitate either protein purification, protein detection orother experimental uses known in the art (3) target secretion ortranslation of a protein to a subcellular organelle, such as theperiplasmic space of Gram-negative bacteria, mitochondria orchloroplasts of plants or the endoplasmic reticulum of eukaryotic cells,the latter of which often results in glycosylation of the protein.

Variant nucleotide and amino acid sequences of the disclosure alsoencompass sequences derived from mutagenic and recombinogenic proceduressuch as DNA shuffling. With such a procedure, one or more differentPtIP-83 polypeptide coding regions can be used to create a new PtIP-83polypeptide possessing the desired properties. In this manner, librariesof recombinant polynucleotides are generated from a population ofrelated sequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between a pesticidal geneand other known pesticidal genes to obtain a new gene coding for aprotein with an improved property of interest, such as an increasedinsecticidal activity. Strategies for such DNA shuffling are known inthe art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer, (1994) Nature 370:389-391; Crameri, et al.,(1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol.272:336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri, et al., (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

Domain swapping or shuffling is another mechanism for generating alteredPtIP-83 polypeptides. Domains may be swapped between PtIP-83polypeptides resulting in hybrid or chimeric toxins with improvedinsecticidal activity or target spectrum. Methods for generatingrecombinant proteins and testing them for pesticidal activity are wellknown in the art (see, for example, Naimov, et al., (2001) Appl.Environ. Microbiol. 67:5328-5330; de Maagd, et al., (1996) Appl.Environ. Microbiol. 62:1537-1543; Ge, et al., (1991) J. Biol. Chem.266:17954-17958; Schnepf, et al., (1990) J. Biol. Chem. 265:20923-20930;Rang, et al., 91999) Appl. Environ. Microbiol. 65:2918-2925).

Alignment of PtIP-83 homologs (FIG. 2) allows for identification ofresidues that are highly conserved among natural homologs in thisfamily.

In some embodiments PtIP-83 polypeptides are provided comprising anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to any one of SEQ ID NO: 786-888.

In some embodiments PtIP-83 polypeptides are provide comprising theamino acid sequence of any one of SEQ ID NO: 786-888.

In some embodiments the PtIP-83 polypeptide is not the amino acidsequence of any one of SEQ ID NO: 786-888.

Compositions

Compositions comprising a PtIP-83 polypeptide of the disclosure are alsoembraced. In some embodiments the composition comprises a PtIP-83polypeptide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ IDNO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758,SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ IDNO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767,SEQ ID NO: 768 or SEQ ID NO: 769 or a variant thereof. In someembodiments the composition comprises a PtIP-83 fusion protein.

In some embodiments compositions are provided comprising a PtIP-83polypeptide comprising an amino acid sequence of any one of SEQ ID NO:236-299, SEQ ID NO: 334-367, SEQ ID NO: 398-427, SEQ ID NO: 518-607, SEQID NO: 640-645, and SEQ ID NO: 728-737 or a variant thereof.

In some embodiments compositions are provide comprising a PtIP-83polypeptide comprising the amino acid sequence of any one of SEQ ID NO:786-888 or a variant thereof.

In some embodiments agricultural compositions of PtIP-83 polypeptidesare disclosed. In the embodiments, a transformed microorganism (whichincludes whole organisms, cells, spore(s), PtIP-83 polypeptide(s),pesticidal component(s), pest-impacting component(s), variant(s), livingor dead cells and cell components, including mixtures of living and deadcells and cell components, and including broken cells and cellcomponents) or an isolated PtIP-83 polypeptide(s) can be formulated withan acceptable carrier into a pesticidal composition(s) that is, forexample, a suspension, a solution, an emulsion, a dusting powder, adispersible granule or pellet, a wettable powder, and an emulsifiableconcentrate, an aerosol or spray, an impregnated granule, an adjuvant, acoatable paste, a colloid, and also encapsulations in, for example,polymer substances. Such formulated compositions may be prepared by suchconventional means as desiccation, lyophilization, homogenization,extraction, filtration, centrifugation, sedimentation, or concentrationof a culture of cells comprising the polypeptide.

Such compositions disclosed above may be obtained by the addition of asurface-active agent, an inert carrier, a preservative, a humectant, afeeding stimulant, an attractant, an encapsulating agent, a binder, anemulsifier, a dye, a UV protectant, a buffer, a flow agent orfertilizers, micronutrient donors, or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bactericides, nematocides,molluscicides, acaricides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular target pests.Suitable carriers and adjuvants can be solid or liquid and correspond tothe substances ordinarily employed in formulation technology, e.g.,natural or regenerated mineral substances, solvents, dispersants,wetting agents, tackifiers, binders, or fertilizers. The activeingredients of the embodiments are normally applied in the form ofcompositions and can be applied to the crop area, plant, or seed to betreated. For example, the compositions of the embodiments may be appliedto grain in preparation for or during storage in a grain bin or silo,etc. The compositions of the embodiments may be applied simultaneouslyor in succession with other compounds. Methods of applying an activeingredient of the embodiments or an agrochemical composition of theembodiments that contains at least one of the CytlA variant polypeptidesproduced by the bacterial strains of the embodiments include, but arenot limited to, foliar application, seed coating, and soil application.The number of applications and the rate of application depend on theintensity of infestation by the corresponding pest.

Suitable surface-active agents include, but are not limited to, anioniccompounds such as a carboxylate of, for example, a metal; a carboxylateof a long chain fatty acid; an N-acylsarcosinate; mono or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecylsulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates;ethoxylated alkylphenol sulfates; lignin sulfonates; petroleumsulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates orlower alkylnaphthalene sulfonates, e.g., butyl-naphthalene sulfonate;salts of sulfonated naphthalene-formaldehyde condensates; salts ofsulfonated phenol-formaldehyde condensates; more complex sulfonates suchas the amide sulfonates, e.g., the sulfonated condensation product ofoleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g.,the sodium sulfonate of dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g., polyoxyethylene sorbitar fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials include but are not limited to inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates, Mica, Amorphous Silica Gel, talc, clay, volcanic ash orbotanical materials such as cork, powdered corncobs, peanut hulls, ricehulls, and walnut shells. Kaolins such as kaolinite, dickite, nacrite,anauxite, halloysite and endellite are useful as carrier materials.Montmorillonites, such as beidellite, nontronite, montmorillonite,hectorite, saponite, sauconite and bentonite are useful as carriermaterials. Vermiculites such as biotite are useful as carrier materials.

The compositions of the embodiments can be in a suitable form for directapplication or as a concentrate of primary composition that requiresdilution with a suitable quantity of water or other diluent beforeapplication. The pesticidal concentration will vary depending upon thenature of the particular formulation, specifically, whether it is aconcentrate or to be used directly. The composition contains 1 to 98% ofa solid or liquid inert carrier, and 0 to 50% or 0.1 to 50% of asurfactant. These compositions will be administered at the labeled ratefor the commercial product, for example, about 0.01 lb-5.0 lb. per acrewhen in dry form and at about 0.01 pts.-10 pts. per acre when in liquidform.

Antibodies

Antibodies to a PtIP-83 polypeptide of the embodiments or to variants orfragments thereof are also encompassed. The antibodies of the disclosureinclude polyclonal and monoclonal antibodies as well as fragmentsthereof which retain their ability to bind to PtIP-83 polypeptide foundin the insect gut. An antibody, monoclonal antibody or fragment thereofis said to be capable of binding a molecule if it is capable ofspecifically reacting with the molecule to thereby bind the molecule tothe antibody, monoclonal antibody or fragment thereof. The term“antibody” (Ab) or “monoclonal antibody” (Mab) is meant to includeintact molecules as well as fragments or binding regions or domainsthereof (such as, for example, Fab and F(ab).sub.2 fragments) which arecapable of binding hapten. Such fragments are typically produced byproteolytic cleavage, such as papain or pepsin. Alternatively,hapten-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry. Methods forthe preparation of the antibodies of the present disclosure aregenerally known in the art. For example, see, Antibodies, A LaboratoryManual, Ed Harlow and David Lane (eds.) Cold Spring Harbor Laboratory,N.Y. (1988), as well as the references cited therein. Standard referenceworks setting forth the general principles of immunology include: Klein,J. Immunology: The Science of Cell-Noncell Discrimination, John Wiley &Sons, N.Y. (1982); Dennett, et al., Monoclonal Antibodies, Hybridoma: ANew Dimension in Biological Analyses, Plenum Press, N.Y. (1980) andCampbell, “Monoclonal Antibody Technology,” In Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 13, Burdon, et al., (eds.),Elsevier, Amsterdam (1984). See also, U.S. Pat. Nos. 4,196,265;4,609,893; 4,713,325; 4,714,681; 4,716,111; 4,716,117 and 4,720,459.PtIP-83 polypeptide antibodies or antigen-binding portions thereof canbe produced by a variety of techniques, including conventionalmonoclonal antibody methodology, for example the standard somatic cellhybridization technique of Kohler and Milstein, (1975) Nature 256:495.Other techniques for producing monoclonal antibody can also be employedsuch as viral or oncogenic transformation of B lymphocytes. An animalsystem for preparing hybridomas is a murine system. Immunizationprotocols and techniques for isolation of immunized splenocytes forfusion are known in the art. Fusion partners (e.g., murine myelomacells) and fusion procedures are also known. The antibody and monoclonalantibodies of the disclosure can be prepared by utilizing a PtIP-83polypeptide as antigens.

A kit for detecting the presence of a PtIP-83 polypeptide or detectingthe presence of a nucleotide sequence encoding a PtIP-83 polypeptide ina sample is provided. In one embodiment, the kit provides antibody-basedreagents for detecting the presence of a PtIP-83 polypeptide in a tissuesample. In another embodiment, the kit provides labeled nucleic acidprobes useful for detecting the presence of one or more polynucleotidesencoding PtIP-83 polypeptide. The kit is provided along with appropriatereagents and controls for carrying out a detection method, as well asinstructions for use of the kit.

Receptor Identification and Isolation

Receptors to the PtIP-83 polypeptide of the embodiments or to variantsor fragments thereof, are also encompassed. Methods for identifyingreceptors are well known in the art (see, Hofmann, et. al., (1988) Eur.J. Biochem. 173:85-91; Gill, et al., (1995) J. Biol. Chem. 27277-27282)can be employed to identify and isolate the receptor that recognizes thePtIP-83 polypeptide using the brush-border membrane vesicles fromsusceptible insects. In addition to the radioactive labeling methodlisted in the cited literatures, PtIP-83 polypeptide can be labeled withfluorescent dye and other common labels such as streptavidin.Brush-border membrane vesicles (BBMV) of susceptible insects such assoybean looper and stink bugs can be prepared according to the protocolslisted in the references and separated on SDS-PAGE gel and blotted onsuitable membrane. Labeled PtIP-83 polypeptide can be incubated withblotted membrane of BBMV and labeled the PtIP-83 polypeptide can beidentified with the labeled reporters. Identification of protein band(s)that interact with the PtIP-83 polypeptide can be detected by N-terminalamino acid gas phase sequencing or mass spectrometry based proteinidentification method (Patterson, (1998) 10.22, 1-24, Current Protocolin Molecular Biology published by John Wiley & Son Inc). Once theprotein is identified, the corresponding gene can be cloned from genomicDNA or cDNA library of the susceptible insects and binding affinity canbe measured directly with the PtIP-83 polypeptide. Receptor function forinsecticidal activity by the PtIP-83 polypeptide can be verified byaccomplished by RNAi type of gene knock out method (Rajagopal, et al.,(2002) J. Biol. Chem. 277:46849-46851).

Nucleotide Constructs, Expression Cassettes and Vectors

The use of the term “nucleotide constructs” herein is not intended tolimit the embodiments to nucleotide constructs comprising DNA. Those ofordinary skill in the art will recognize that nucleotide constructsparticularly polynucleotides and oligonucleotides composed ofribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides may also be employed in the methods disclosedherein. The nucleotide constructs, nucleic acids, and nucleotidesequences of the embodiments additionally encompass all complementaryforms of such constructs, molecules, and sequences. Further, thenucleotide constructs, nucleotide molecules, and nucleotide sequences ofthe embodiments encompass all nucleotide constructs, molecules, andsequences which can be employed in the methods of the embodiments fortransforming plants including, but not limited to, those comprised ofdeoxyribonucleotides, ribonucleotides, and combinations thereof. Suchdeoxyribonucleotides and ribonucleotides include both naturallyoccurring molecules and synthetic analogues. The nucleotide constructs,nucleic acids, and nucleotide sequences of the embodiments alsoencompass all forms of nucleotide constructs including, but not limitedto, single-stranded forms, double-stranded forms, hairpins,stem-and-loop structures and the like.

A further embodiment relates to a transformed organism such as anorganism selected from plant and insect cells, bacteria, yeast,baculovirus, protozoa, nematodes and algae. The transformed organismcomprises a DNA molecule of the embodiments, an expression cassettecomprising the DNA molecule or a vector comprising the expressioncassette, which may be stably incorporated into the genome of thetransformed organism.

In some embodiments transgenic host cells are provide transformed with apolynucleotide encoding a PtIP-83 polypeptide of the disclosure. In someembodiments the host cell is a plant cell. In some embodiments the hostcell is a bacteria.

The sequences of the embodiments are provided in DNA constructs forexpression in the organism of interest. The construct will include 5′and 3′ regulatory sequences operably linked to a sequence of theembodiments. The term “operably linked” as used herein refers to afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand where necessary to join two protein coding regions in the samereading frame. The construct may additionally contain at least oneadditional gene to be cotransformed into the organism. Alternatively,the additional gene(s) can be provided on multiple DNA constructs.

Such a DNA construct is provided with a plurality of restriction sitesfor insertion of the PtIP-83 polypeptide gene sequence to be under thetranscriptional regulation of the regulatory regions. The DNA constructmay additionally contain selectable marker genes.

The DNA construct will generally include in the 5′ to 3′ direction oftranscription: a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the embodiments, and atranscriptional and translational termination region (i.e., terminationregion) functional in the organism serving as a host. Thetranscriptional initiation region (i.e., the promoter) may be native,analogous, foreign or heterologous to the host organism and/or to thesequence of the embodiments. Additionally, the promoter may be thenatural sequence or alternatively a synthetic sequence. The term“foreign” as used herein indicates that the promoter is not found in thenative organism into which the promoter is introduced. Where thepromoter is “foreign” or “heterologous” to the sequence of theembodiments, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked sequence of theembodiments. As used herein, a chimeric gene comprises a coding sequenceoperably linked to a transcription initiation region that isheterologous to the coding sequence. Where the promoter is a native ornatural sequence, the expression of the operably linked sequence isaltered from the wild-type expression, which results in an alteration inphenotype.

In some embodiments the DNA construct may also include a transcriptionalenhancer sequence. As used herein, the term an “enhancer” refers to aDNA sequence which can stimulate promoter activity, and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Various enhancers areknown in the art including for example, introns with gene expressionenhancing properties in plants (US Patent Application Publication Number2009/0144863, the ubiquitin intron (i.e., the maize ubiquitin intron 1(see, for example, NCBI sequence S94464)), the omega enhancer or theomega prime enhancer (Gallie, et al., (1989) Molecular Biology of RNAed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, et al., (1990)EMBO J. 9:1685-96) and the enhancers of U.S. Pat. No. 7,803,992 may alsobe used, each of which is incorporated by reference. The above list oftranscriptional enhancers is not meant to be limiting. Any appropriatetranscriptional enhancer can be used in the embodiments.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host or may be derived from another source(i.e., foreign or heterologous to the promoter, the sequence ofinterest, the plant host or any combination thereof).

Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau, et al., (1991) Mol. Gen.Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al.,(1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al.,(1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al., (1987) NucleicAcid Res. 15:9627-9639.

Where appropriate, a nucleic acid may be optimized for increasedexpression in the host organism. Thus, where the host organism is aplant, the synthetic nucleic acids can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for a discussion ofhost-preferred codon usage. For example, although nucleic acid sequencesof the embodiments may be expressed in both monocotyledonous anddicotyledonous plant species, sequences can be modified to account forthe specific codon preferences and GC content preferences ofmonocotyledons or dicotyledons as these preferences have been shown todiffer (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, themaize-preferred codon for a particular amino acid may be derived fromknown gene sequences from maize. Maize codon usage for 28 genes frommaize plants is listed in Table 4 of Murray, et al., supra. Methods areavailable in the art for synthesizing plant-preferred genes. See, forexample, U.S. Pat. Nos. 5,380,831, and 5,436,391 and Murray, et al.,(1989) Nucleic Acids Res. 17:477-498, and Liu H et al. Mol Bio Rep37:677-684, 2010, herein incorporated by reference. A Zea maize codonusage table can be also found atkazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=4577, which can beaccessed using the www prefix. Table 3 shows a maize optimal codonanalysis (adapted from Liu H et al. Mol Bio Rep 37:677-684, 2010).

TABLE 3 Amino High Low Amino High Low Acid Codon Count RSCU Count RSCUAcid Codon Count RSCU Count RSCU Phe UUU 115 0.04 2,301 1.22 Ala GCU 6290.17 3,063 1.59 UUC* 5,269 1.96 1,485 0.78 GCC* 8,057 2.16 1,136 0.59Ser UCU 176 0.13 2,498 1.48 GCA 369 0.1 2,872 1.49 UCC* 3,489 2.48 1,0740.63 GCG* 5,835 1.57 630 0.33 UCA 104 0.07 2,610 1.54 Tyr UAU 71 0.041,632 1.22 UCG* 1,975 1.4 670 0.4 UAC* 3,841 1.96 1,041 0.78 AGU 77 0.051,788 1.06 His CAU 131 0.09 1,902 1.36 AGC* 2,617 1.86 1,514 0.89 CAC*2,800 1.91 897 0.64 Leu UUA 10 0.01 1,326 0.79 Cys UGU 52 0.04 1,2331.12 UUG 174 0.09 2,306 1.37 UGC* 2,291 1.96 963 0.88 CUU 223 0.11 2,3961.43 Gln CAA 99 0.05 2,312 1.04 CUC* 5,979 3.08 1,109 0.66 CAG* 3,5571.95 2,130 0.96 CUA 106 0.05 1,280 0.76 Arg CGU 153 0.12 751 0.74 CUG*5,161 2.66 1,646 0.98 CGC* 4,278 3.25 466 0.46 Pro CCU 427 0.22 1,9001.47 CGA 92 0.07 659 0.65 CCC* 3,035 1.59 601 0.47 CGG* 1,793 1.36 6310.62 CCA 311 0.16 2,140 1.66 AGA 83 0.06 1,948 1.91 CCG* 3,846 2.02 5130.4 AGG* 1,493 1.14 1,652 1.62 Ile AUU 138 0.09 2,388 1.3 Asn AAU 1310.07 3,074 1.26 AUC* 4,380 2.85 1,353 0.74 AAC* 3,814 1.93 1,807 0.74AUA 88 0.06 1,756 0.96 Lys AAA 130 0.05 3,215 0.98 Thr ACU 136 0.091,990 1.43 AAG* 5,047 1.95 3,340 1.02 ACC* 3,398 2.25 991 0.71 Asp GAU312 0.09 4,217 1.38 ACA 133 0.09 2,075 1.5 GAC* 6,729 1.91 1,891 0.62ACG* 2,378 1.57 495 0.36 Gly GGU 363 0.13 2,301 1.35 Val GUU 182 0.072,595 1.51 GGC* 7,842 2.91 1,282 0.75 GUC* 4,584 1.82 1,096 0.64 GGA 3970.15 2,044 1.19 GUA 74 0.03 1,325 0.77 GGG* 2,186 0.81 1,215 0.71 GUG*5,257 2.08 1,842 1.07 Glu GAA 193 0.06 4,080 1.1 GAG* 6,010 1.94 3,3070.9 Codon usage was compared using Chi squared contingency test toidentify optimal codons. Codons that occur significantly more often(P\0.01) are indicated with an asterisk.

TABLE 4 TTT F 21.2 (10493) TCT S 18.4 (9107) TTC F 21.2 (10487) TCC S12.9 (6409) TTA L 9.2  (4545) TCA S 15.6 (7712) TTG L 22.9 (11340) TCG S4.8 (2397) CTT L 23.9 (11829) CCT P 18.9 (9358) CTC L 17.1  (8479) CCC P10.1 (5010) CTA L 8.5  (4216) CCA P 19.1 (9461) CTG L 12.7  (6304) CCG P4.7 (2312) ATT I 25.1 (12411) ACT T 17.1 (8490) ATC I 16.3  (8071) ACC T14.3 (7100) ATA I 12.9  (6386) ACA T 14.9 (7391) ATG M 22.7 (11218) ACGT 4.3 (2147) GTT V 26.1 (12911) GCT A 26.7 (13201)  GTC V 11.9  (5894)GCC A 16.2 (8026) GTA V 7.7  (3803) GCA A 21.4 (10577)  GTG V 21.4(10610) GCG A 6.3 (3123) TAT Y 15.7  (7779) TGT C 8.1 (3995) TAC Y 14.9 (7367) TGC C 8.0 (3980) TAA * 0.9   (463) TGA * 1.0  (480) TAG * 0.5  (263) TGG W 13.0 (6412) CAT H 14.0  (6930) CGT R 6.6 (3291) CAC H 11.6 (5759) CGC R 6.2 (3093) CAA Q 20.5 (10162) CGA R 4.1 (2018) CAG Q 16.2 (8038) CGG R 3.1 (1510) AAT N 22.4 (11088) AGT S 12.6 (6237) AAC N 22.8(11284) AGC S 11.3 (5594) AAA K 26.9 (13334) AGA R 14.8 (7337) AAG K35.9 (17797) AGG R 13.3 (6574) GAT D 32.4 (16040) GGT G 20.9 (10353) GAC D 20.4 (10097) GGC G 13.4 (6650) GAA E 33.2 (16438) GGA G 22.3(11022)  GAG E 33.2 (16426) GGG G 13.0 (6431)

A Glycine max codon usage table is shown in Table 4 and can also befound atkazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=3847&aa=1&style=N,which can be accessed using the www prefix.

In some embodiments the recombinant nucleic acid molecule encoding aPtIP-83 polypeptide has maize optimized codons.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other well-characterized sequences that maybe deleterious to gene expression. The GC content of the sequence may beadjusted to levels average for a given cellular host, as calculated byreference to known genes expressed in the host cell. The term “hostcell” as used herein refers to a cell which contains a vector andsupports the replication and/or expression of the expression vector isintended. Host cells may be prokaryotic cells such as E. coli oreukaryotic cells such as yeast, insect, amphibian or mammalian cells ormonocotyledonous or dicotyledonous plant cells. An example of amonocotyledonous host cell is a maize host cell. When possible, thesequence is modified to avoid PtIP-hairpin secondary mRNA structures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie,et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus), human immunoglobulin heavy-chain binding protein (BiP) (Macejak,et al., (1991) Nature 353:90-94); untranslated leader from the coatprotein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al.,(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie,et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York),pp. 237-256) and maize chlorotic mottle virus leader (MCMV) (Lommel, etal., (1991) Virology 81:382-385). See also, Della-Cioppa, et al., (1987)Plant Physiol. 84:965-968. Such constructs may also contain a “signalsequence” or “leader sequence” to facilitate co-translational orpost-translational transport of the peptide to certain intracellularstructures such as the chloroplast (or other plastid), endoplasmicreticulum or Golgi apparatus.

“Signal sequence” as used herein refers to a sequence that is known orsuspected to result in cotranslational or post-translational peptidetransport across the cell membrane. In eukaryotes, this typicallyinvolves secretion into the Golgi apparatus, with some resultingglycosylation. Insecticidal toxins of bacteria are often synthesized asprotoxins, which are protolytically activated in the gut of the targetpest (Chang, (1987) Methods Enzymol. 153:507-516). In some embodiments,the signal sequence is located in the native sequence or may be derivedfrom a sequence of the embodiments. “Leader sequence” as used hereinrefers to any sequence that when translated, results in an amino acidsequence sufficient to trigger co-translational transport of the peptidechain to a subcellular organelle. Thus, this includes leader sequencestargeting transport and/or glycosylation by passage into the endoplasmicreticulum, passage to vacuoles, plastids including chloroplasts,mitochondria, and the like. Nuclear-encoded proteins targeted to thechloroplast thylakoid lumen compartment have a characteristic bipartitetransit peptide, composed of a stromal targeting signal peptide and alumen targeting signal peptide. The stromal targeting information is inthe amino-proximal portion of the transit peptide. The lumen targetingsignal peptide is in the carboxyl-proximal portion of the transitpeptide, and contains all the information for targeting to the lumen.Recent research in proteomics of the higher plant chloroplast hasachieved in the identification of numerous nuclear-encoded lumenproteins (Kieselbach et al. FEBS LETT 480:271-276, 2000; Peltier et al.Plant Cell 12:319-341, 2000; Bricker et al. Biochim. Biophys Acta1503:350-356, 2001), the lumen targeting signal peptide of which canpotentially be used in accordance with the present disclosure. About 80proteins from Arabidopsis, as well as homologous proteins from spinachand garden pea, are reported by Kieselbach et al., PhotosynthesisResearch, 78:249-264, 2003. In particular, Table 2 of this publication,which is incorporated into the description herewith by reference,discloses 85 proteins from the chloroplast lumen, identified by theiraccession number (see also US Patent Application Publication2009/09044298). In addition, the recently published draft version of therice genome (Goff et al, Science 296:92-100, 2002) is a suitable sourcefor lumen targeting signal peptide which may be used in accordance withthe present disclosure.

Suitable chloroplast transit peptides (CTP) are well known to oneskilled in the art also include chimeric CTPs comprising but not limitedto, an N-terminal domain, a central domain or a C-terminal domain from aCTP from Oryza sativa 1-deoxy-D xyulose-5-Phosphate Synthase Oryzasativa-Superoxide dismutase Oryza sativa-soluble starch synthase Oryzasativa-NADP-dependent Malic acid enzyme Oryzasativa-Phospho-2-dehydro-3-deoxyheptonate Aldolase 2 Oryzasativa-L-Ascorbate peroxidase 5 Oryza sativa-Phosphoglucan waterdikinase, Zea Mays ssRUBISCO, Zea Mays-beta-glucosidase, Zea Mays-Malatedehydrogenase, Zea Mays Thioredoxin M-type US Patent ApplicationPublication 2012/0304336).

The PtIP-83 polypeptide gene to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin codon usage between the plant nucleus and this organelle. In thismanner, the nucleic acids of interest may be synthesized usingchloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,herein incorporated by reference.

In preparing the expression cassette, the various DNA fragments may bemanipulated so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used in the practice of the embodiments.The promoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-preferred, inducible orother promoters for expression in the host organism. Suitableconstitutive promoters for use in a plant host cell include, forexample, the core promoter of the Rsyn7 promoter and other constitutivepromoters disclosed in WO 1999/43838 and U.S. Pat. No. 6,072,050; thecore CaMV 35S promoter (Odell, et al., (1985) Nature 313:810-812); riceactin (McElroy, et al., (1990) Plant Cell 2:163-171); ubiquitin(Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 andChristensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, etal., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1984)EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026) and thelike. Other constitutive promoters include, for example, those discussedin U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142 and 6,177,611.

Depending on the desired outcome, it may be beneficial to express thegene from an inducible promoter. Of particular interest for regulatingthe expression of the nucleotide sequences of the embodiments in plantsare wound-inducible promoters. Such wound-inducible promoters, mayrespond to damage caused by insect feeding, and include potatoproteinase inhibitor (pin II) gene (Ryan, (1990) Ann. Rev. Phytopath.28:425-449; Duan, et al., (1996) Nature Biotechnology 14:494-498); wun1and wun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford, et al.,(1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl, et al., (1992)Science 225:1570-1573); WIP1 (Rohmeier, et al., (1993) Plant Mol. Biol.22:783-792; Eckelkamp, et al., (1993) FEBS Letters 323:73-76); MPI gene(Corderok, et al., (1994) Plant J. 6(2):141-150) and the like, hereinincorporated by reference.

Additionally, pathogen-inducible promoters may be employed in themethods and nucleotide constructs of the embodiments. Suchpathogen-inducible promoters include those from pathogenesis-relatedproteins (PR proteins), which are induced following infection by apathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,chitinase, etc. See, for example, Redolfi, et al., (1983) Neth. J. PlantPathol. 89:245-254; Uknes, et al., (1992) Plant Cell 4: 645-656 and VanLoon, (1985) Plant Mol. Virol. 4:111-116. See also, WO 1999/43819,herein incorporated by reference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau, et al., (1987) PlantMol. Biol. 9:335-342; Matton, et al., (1989) Molecular Plant—MicrobeInteractions 2:325-331; Somsisch, et al., (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. 2:93-98 andYang, (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen,et al., (1996) Plant J. 10:955-966; Zhang, et al., (1994) Proc. Natl.Acad. Sci. USA 91:2507-2511; Warner, et al., (1993) Plant J. 3:191-201;Siebertz, et al., (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible) and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero, et al., (1992) Physiol. Mol. Plant Path. 41:189-200).

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression or a chemical-repressible promoter, where application ofthe chemical represses gene expression. Chemical-inducible promoters areknown in the art and include, but are not limited to, the maize In2-2promoter, which is activated by benzenesulfonamide herbicide safeners,the maize GST promoter, which is activated by hydrophobic electrophiliccompounds that are used as pre-emergent herbicides, and the tobaccoPR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena, et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis, et al., (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237 and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced PtIP-83polypeptide expression within a particular plant tissue.Tissue-preferred promoters include those discussed in Yamamoto, et al.,(1997) Plant J. 12(2)255-265; Kawamata, et al., (1997) Plant CellPhysiol. 38(7):792-803; Hansen, et al., (1997) Mol. Gen Genet.254(3):337-343; Russell, et al., (1997) Transgenic Res. 6(2):157-168;Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1341; Van Camp, etal., (1996) Plant Physiol. 112(2):525-535; Canevascini, et al., (1996)Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994) Plant CellPhysiol. 35(5):773-778; Lam, (1994) Results Probl. Cell Differ.20:181-196; Orozco, et al., (1993) Plant Mol Biol. 23(6):1129-1138;Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 andGuevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Such promoters canbe modified, if necessary, for weak expression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kwon, et al., (1994)Plant Physiol. 105:357-67; Yamamoto, et al., (1994) Plant Cell Physiol.35(5):773-778; Gotor, et al., (1993) Plant J. 3:509-18; Orozco, et al.,(1993) Plant Mol. Biol. 23(6):1129-1138 and Matsuoka, et al., (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred or root-specific promoters are known and can be selectedfrom the many available from the literature or isolated de novo fromvarious compatible species. See, for example, Hire, et al., (1992) PlantMol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetasegene); Keller and Baumgartner, (1991) Plant Cell 3(10):1051-1061(root-specific control element in the GRP 1.8 gene of French bean);Sanger, et al., (1990) Plant Mol. Biol. 14(3):433-443 (root-specificpromoter of the mannopine synthase (MAS) gene of Agrobacteriumtumefaciens) and Miao, et al., (1991) Plant Cell 3(1):11-22 (full-lengthcDNA clone encoding cytosolic glutamine synthetase (GS), which isexpressed in roots and root nodules of soybean). See also, Bogusz, etal., (1990) Plant Cell 2(7):633-641, where two root-specific promotersisolated from hemoglobin genes from the nitrogen-fixing nonlegumeParasponia andersonii and the related non-nitrogen-fixing nonlegumeTrema tomentosa are described. The promoters of these genes were linkedto a 3-glucuronidase reporter gene and introduced into both thenonlegume Nicotiana tabacum and the legume Lotus corniculatus, and inboth instances root-specific promoter activity was preserved. Leach andAoyagi, (1991) describe their analysis of the promoters of the highlyexpressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes(see, Plant Science (Limerick) 79(1):69-76). They concluded thatenhancer and tissue-preferred DNA determinants are dissociated in thosepromoters. Teeri, et al., (1989) used gene fusion to lacZ to show thatthe Agrobacterium T-DNA gene encoding octopine synthase is especiallyactive in the epidermis of the root tip and that the TR2′ gene is rootspecific in the intact plant and stimulated by wounding in leaf tissue,an especially desirable combination of characteristics for use with aninsecticidal or larvicidal gene (see, EMBO J. 8(2):343-350). The TR1′gene fused to nptII (neomycin phosphotransferase II) showed similarcharacteristics. Additional root-preferred promoters include theVfENOD-GRP3 gene promoter (Kuster, et al., (1995) Plant Mol. Biol.29(4):759-772) and rolB promoter (Capana, et al., (1994) Plant Mol.Biol. 25(4):681-691. See also, U.S. Pat. Nos. 5,837,876; 5,750,386;5,633,363; 5,459,252; 5,401,836; 5,110,732 and 5,023,179. Arabidopsisthaliana root-preferred regulatory sequences are disclosed inUS20130117883.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See, Thompson, et al., (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); and milps(myo-inositol-1-phosphate synthase) (see, U.S. Pat. No. 6,225,529,herein incorporated by reference). Gamma-zein and Glb-1 areendosperm-specific promoters. For dicots, seed-specific promotersinclude, but are not limited to, Kunitz trypsin inhibitor 3 (KTi3)(Jofuku and Goldberg, (1989) Plant Cell 1:1079-1093), bean β-phaseolin,napin, β-conglycinin, glycinin 1, soybean lectin, cruciferin, and thelike. For monocots, seed-specific promoters include, but are not limitedto, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken1, shrunken 2, globulin 1, etc. See also, WO 2000/12733, whereseed-preferred promoters from end1 and end2 genes are disclosed; hereinincorporated by reference. In dicots, seed specific promoters includebut are not limited to seed coat promoter from Arabidopsis, pBAN; andthe early seed promoters from Arabidopsis, p26, p63, and p63tr (U.S.Pat. Nos. 7,294,760 and 7,847,153). A promoter that has “preferred”expression in a particular tissue is expressed in that tissue to agreater degree than in at least one other plant tissue. Sometissue-preferred promoters show expression almost exclusively in theparticular tissue.

Where low level expression is desired, weak promoters will be used.Generally, the term “weak promoter” as used herein refers to a promoterthat drives expression of a coding sequence at a low level. By low levelexpression at levels of between about 1/1000 transcripts to about1/100,000 transcripts to about 1/500,000 transcripts is intended.Alternatively, it is recognized that the term “weak promoters” alsoencompasses promoters that drive expression in only a few cells and notin others to give a total low level of expression. Where a promoterdrives expression at unacceptably high levels, portions of the promotersequence can be deleted or modified to decrease expression levels.

Such weak constitutive promoters include, for example the core promoterof the Rsyn7 promoter (WO 1999/43838 and U.S. Pat. No. 6,072,050), thecore 35S CaMV promoter, and the like. Other constitutive promotersinclude, for example, those disclosed in U.S. Pat. Nos. 5,608,149;5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;5,608,142 and 6,177,611, herein incorporated by reference.The above list of promoters is not meant to be limiting. Any appropriatepromoter can be used in the embodiments.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glufosinate ammonium, bromoxynil, imidazolinones and2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitableselectable marker genes include, but are not limited to, genes encodingresistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J.2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature303:209-213 and Meijer, et al., (1991) Plant Mol. Biol. 16:807-820);streptomycin (Jones, et al., (1987) Mol. Gen. Genet. 210:86-91);spectinomycin (Bretagne-Sagnard, et al., (1996) Transgenic Res.5:131-137); bleomycin (Hille, et al., (1990) Plant Mol. Biol.7:171-176); sulfonamide (Guerineau, et al., (1990) Plant Mol. Biol.15:127-136); bromoxynil (Stalker, et al., (1988) Science 242:419-423);glyphosate (Shaw, et al., (1986) Science 233:478-481 and U.S. patentapplication Ser. Nos. 10/004,357 and 10/427,692); phosphinothricin(DeBlock, et al., (1987) EMBO J. 6:2513-2518). See generally, Yarranton,(1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et al., (1992)Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao, et al., (1992) Cell71:63-72; Reznikoff, (1992) Mol. Microbiol. 6:2419-2422; Barkley, etal., (1980) in The Operon, pp. 177-220; Hu, et al., (1987) Cell48:555-566; Brown, et al., (1987) Cell 49:603-612; Figge, et al., (1988)Cell 52:713-722; Deuschle, et al., (1989) Proc. Natl. Acad. Sci. USA86:5400-5404; Fuerst, et al., (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle, et al., (1990) Science 248:480-483; Gossen,(1993) Ph.D. Thesis, University of Heidelberg; Reines, et al., (1993)Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) Mol.Cell. Biol. 10:3343-3356; Zambretti, et al., (1992) Proc. Natl. Acad.Sci. USA 89:3952-3956; Baim, et al., (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10:143-162;Degenkolb, et al., (1991) Antimicrob. Agents Chemother. 35:1591-1595;Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; Bonin, (1993)Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc.Natl. Acad. Sci. USA 89:5547-5551; Oliva, et al., (1992) Antimicrob.Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook ofExperimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin) and Gill,et al., (1988) Nature 334:721-724. Such disclosures are hereinincorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the embodiments.

Plant Transformation

The methods of the embodiments involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is as used herein meanspresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the embodiments do not depend on a particularmethod for introducing a polynucleotide or polypeptide into a plant,only that the polynucleotide or polypeptides gains access to theinterior of at least one cell of the plant. Methods for introducingpolynucleotide or polypeptides into plants are known in the artincluding, but not limited to, stable transformation methods, transienttransformation methods, and virus-mediated methods.

“Stable transformation” is as used herein means that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” as used herein means that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant. “Plant” as usedherein refers to whole plants, plant organs (e.g., leaves, stems, roots,etc.), seeds, plant cells, propagules, embryos and progeny of the same.Plant cells can be differentiated or undifferentiated (e.g. callus,suspension culture cells, protoplasts, leaf cells, root cells, phloemcells and pollen).

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway, et al., (1986) Biotechniques 4:320-334), electroporation(Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606),Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and5,981,840), direct gene transfer (Paszkowski, et al., (1984) EMBO J.3:2717-2722) and ballistic particle acceleration (see, for example, U.S.Pat. Nos. 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes, et al.,(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods,ed. Gamborg and Phillips, (Springer-Verlag, Berlin) and McCabe, et al.,(1988) Biotechnology 6:923-926) and Lecl transformation (WO 00/28058).For potato transformation see, Tu, et al., (1998) Plant MolecularBiology 37:829-838 and Chong, et al., (2000) Transgenic Research9:71-78. Additional transformation procedures can be found inWeissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al.,(1987) Particulate Science and Technology 5:27-37 (onion); Christou, etal., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor.Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563(maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein, etal., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984)Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals);Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman, et al., (Longman, New York), pp. 197-209(pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 andKaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413(rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maizevia Agrobacterium tumefaciens); all of which are herein incorporated byreference.

In specific embodiments, the sequences of the embodiments can beprovided to a plant using a variety of transient transformation methods.Such transient transformation methods include, but are not limited to,the introduction of the PtIP-83 polynucleotide or variants and fragmentsthereof directly into the plant or the introduction of the PtIP-83polypeptide transcript into the plant. Such methods include, forexample, microinjection or particle bombardment. See, for example,Crossway, et al., (1986) Mol Gen. Genet. 202:179-185; Nomura, et al.,(1986) Plant Sci. 44:53-58; Hepler, et al., (1994) Proc. Natl. Acad.Sci. 91:2176-2180 and Hush, et al., (1994) The Journal of Cell Science107:775-784, all of which are herein incorporated by reference.Alternatively, the PtIP-83 polypeptide polynucleotide can be transientlytransformed into the plant using techniques known in the art. Suchtechniques include viral vector system and the precipitation of thepolynucleotide in a manner that precludes subsequent release of the DNA.Thus, transcription from the particle-bound DNA can occur, but thefrequency with which it is released to become integrated into the genomeis greatly reduced. Such methods include the use of particles coatedwith polyethylimine (PEI; Sigma # P3143).

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO 1999/25855and WO 1999/25853, all of which are herein incorporated by reference.Briefly, the polynucleotide of the embodiments can be contained intransfer cassette flanked by two non-identical recombination sites. Thetransfer cassette is introduced into a plant have stably incorporatedinto its genome a target site which is flanked by two non-identicalrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome.

Plant transformation vectors may be comprised of one or more DNA vectorsneeded for achieving plant transformation. For example, it is a commonpractice in the art to utilize plant transformation vectors that arecomprised of more than one contiguous DNA segment. These vectors areoften referred to in the art as “binary vectors”. Binary vectors as wellas vectors with helper plasmids are most often used forAgrobacterium-mediated transformation, where the size and complexity ofDNA segments needed to achieve efficient transformation is quite large,and it is advantageous to separate functions onto separate DNAmolecules. Binary vectors typically contain a plasmid vector thatcontains the cis-acting sequences required for T-DNA transfer (such asleft border and right border), a selectable marker that is engineered tobe capable of expression in a plant cell, and a “gene of interest” (agene engineered to be capable of expression in a plant cell for whichgeneration of transgenic plants is desired). Also present on thisplasmid vector are sequences required for bacterial replication. Thecis-acting sequences are arranged in a fashion to allow efficienttransfer into plant cells and expression therein. For example, theselectable marker gene and the pesticidal gene are located between theleft and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as is understood in the art (Hellens and Mullineaux, (2000)Trends in Plant Science 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethylene glycol, etc.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g., immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Following integration of heterologous foreign DNA into plant cells, onethen applies a maximum threshold level of appropriate selection in themedium to kill the untransformed cells and separate and proliferate theputatively transformed cells that survive from this selection treatmentby transferring regularly to a fresh medium. By continuous passage andchallenge with appropriate selection, one identifies and proliferatesthe cells that are transformed with the plasmid vector. Molecular andbiochemical methods can then be used to confirm the presence of theintegrated heterologous gene of interest into the genome of thetransgenic plant.

Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grows into amature plant and produces fertile seeds (e.g., Hiei, et al., (1994) ThePlant Journal 6:271-282; Ishida, et al., (1996) Nature Biotechnology14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plants are found inAyres and Park, (1994) Critical Reviews in Plant Science 13:219-239 andBommineni and Jauhar, (1997) Maydica 42:107-120. Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick, et al.,(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive or inducible expression ofthe desired phenotypic characteristic identified. Two or moregenerations may be grown to ensure that expression of the desiredphenotypic characteristic is stably maintained and inherited and thenseeds harvested to ensure that expression of the desired phenotypiccharacteristic has been achieved.

The nucleotide sequences of the embodiments may be provided to the plantby contacting the plant with a virus or viral nucleic acids. Generally,such methods involve incorporating the nucleotide construct of interestwithin a viral DNA or RNA molecule. It is recognized that therecombinant proteins of the embodiments may be initially synthesized aspart of a viral polyprotein, which later may be processed by proteolysisin vivo or in vitro to produce the desired PtIP-83 polypeptide. It isalso recognized that such a viral polyprotein, comprising at least aportion of the amino acid sequence of a PtIP-83 of the embodiments, mayhave the desired pesticidal activity. Such viral polyproteins and thenucleotide sequences that encode for them are encompassed by theembodiments. Methods for providing plants with nucleotide constructs andproducing the encoded proteins in the plants, which involve viral DNA orRNA molecules are known in the art. See, for example, U.S. Pat. Nos.5,889,191; 5,889,190; 5,866,785; 5,589,367 and 5,316,931; hereinincorporated by reference.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab, et al., (1990) Proc. Natl. Acad. Sci. USA87:8526-8530; Svab and Maliga, (1993) Proc. Natl. Acad. Sci. USA90:913-917; Svab and Maliga, (1993) EMBO J. 12:601-606. The methodrelies on particle gun delivery of DNA containing a selectable markerand targeting of the DNA to the plastid genome through homologousrecombination. Additionally, plastid transformation can be accomplishedby transactivation of a silent plastid-borne transgene bytissue-preferred expression of a nuclear-encoded and plastid-directedRNA polymerase. Such a system has been reported in McBride, et al.,(1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

The embodiments further relate to plant-propagating material of atransformed plant of the embodiments including, but not limited to,seeds, tubers, corms, bulbs, leaves and cuttings of roots and shoots.

The embodiments may be used for transformation of any plant species,including, but not limited to, monocots and dicots. Examples of plantsof interest include, but are not limited to, corn (Zea mays), Brassicasp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers that may beemployed in practicing the embodiments include, for example, pines suchas loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosapine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Montereypine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Westernhemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Plants of the embodiments include crop plants (for example, corn,alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut,sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

Turf grasses include, but are not limited to: annual bluegrass (Poaannua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poacompressa); Chewing's fescue (Festuca rubra); colonial bentgrass(Agrostis tenuis); creeping bentgrass (Agrostis palustris); crestedwheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyroncristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poapratensis); orchardgrass (Dactylis glomerata); perennial ryegrass(Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba);rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smoothbromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy(Phleum pratense); velvet bentgrass (Agrostis canina); weepingalkaligrass (Puccinellia distans); western wheatgrass (Agropyronsmithii); Bermuda grass (Cynodon spp.); St. Augustine grass(Stenotaphrum secundatum); zoysia grass (Zoysia spp.); Bahia grass(Paspalum notatum); carpet grass (Axonopus affinis); centipede grass(Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum);seashore paspalum (Paspalum vaginatum); blue gramma (Boutelouagracilis); buffalo grass (Buchloe dactyloids); sideoats gramma(Bouteloua curtipendula).

Plants of interest include grain plants that provide seeds of interest,oil-seed plants, and leguminous plants. Seeds of interest include grainseeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc.Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica,maize, alfalfa, palm, coconut, flax, castor, olive, etc. Leguminousplants include beans and peas. Beans include guar, locust bean,fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, favabean, lentils, chickpea, etc.

Evaluation of Plant Transformation

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of heterologous gene in the plant genomeis confirmed by various methods such as analysis of nucleic acids,proteins and metabolites associated with the integrated gene. PCRanalysis is a rapid method to screen transformed cells, tissue or shootsfor the presence of incorporated gene at the earlier stage beforetransplanting into the soil (Sambrook and Russell, (2001) MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, (2001) supra). In general, total DNAis extracted from the transformant, digested with appropriaterestriction enzymes, fractionated in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” is then probedwith, for example, radiolabeled 32P target DNA fragment to confirm theintegration of introduced gene into the plant genome according tostandard techniques (Sambrook and Russell, (2001) supra).

In Northern blot analysis, RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, and blottedonto a nylon filter according to standard procedures that are routinelyused in the art (Sambrook and Russell, (2001) supra). Expression of RNAencoded by the pesticidal gene is then tested by hybridizing the filterto a radioactive probe derived from a pesticidal gene, by methods knownin the art (Sambrook and Russell, (2001) supra).

Western blot, biochemical assays and the like may be carried out on thetransgenic plants to confirm the presence of protein encoded by thepesticidal gene by standard procedures (Sambrook and Russell, 2001,supra) using antibodies that bind to one or more epitopes present on thePtIP-83 polypeptide.

Stacking of Traits in Transgenic Plant

Transgenic plants may comprise a stack of one or more insecticidalpolynucleotides disclosed herein with one or more additionalpolynucleotides resulting in the production or suppression of multiplepolypeptide sequences. Transgenic plants comprising stacks ofpolynucleotide sequences can be obtained by either or both oftraditional breeding methods or through genetic engineering methods.These methods include, but are not limited to, breeding individual lineseach comprising a polynucleotide of interest, transforming a transgenicplant comprising a gene disclosed herein with a subsequent gene andco-transformation of genes into a single plant cell. As used herein, theterm “stacked” includes having the multiple traits present in the sameplant (i.e., both traits are incorporated into the nuclear genome, onetrait is incorporated into the nuclear genome and one trait isincorporated into the genome of a plastid or both traits areincorporated into the genome of a plastid). In one non-limiting example,“stacked traits” comprise a molecular stack where the sequences arephysically adjacent to each other. A trait, as used herein, refers tothe phenotype derived from a particular sequence or groups of sequences.Co-transformation of genes can be carried out using singletransformation vectors comprising multiple genes or genes carriedseparately on multiple vectors. If the sequences are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. The traits can beintroduced simultaneously in a co-transformation protocol with thepolynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO1999/25855 and WO 1999/25853, all of which are herein incorporated byreference.

In some embodiments the polynucleotides encoding the PtIP-83 polypeptidedisclosed herein, alone or stacked with one or more additional insectresistance traits can be stacked with one or more additional inputtraits (e.g., herbicide resistance, fungal resistance, virus resistance,stress tolerance, disease resistance, male sterility, stalk strength,and the like) or output traits (e.g., increased yield, modifiedstarches, improved oil profile, balanced amino acids, high lysine ormethionine, increased digestibility, improved fiber quality, droughtresistance, and the like). Thus, the polynucleotide embodiments can beused to provide a complete agronomic package of improved crop qualitywith the ability to flexibly and cost effectively control any number ofagronomic pests.

Transgenes useful for stacking include but are not limited to:

1. Transgenes that Confer Resistance to Insects or Disease and thatEncode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Jones, et al., (1994) Science266:789 (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfulvum); Martin, et al., (1993) Science 262:1432 (tomato Pto gene forresistance to Pseudomonas syringae pv. tomato encodes a protein kinase);Mindrinos, et al., (1994) Cell 78:1089 (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae), McDowell and Woffenden, (2003)Trends Biotechnol. 21(4):178-83 and Toyoda, et al., (2002) TransgenicRes. 11 (6):567-82. A plant resistant to a disease is one that is moreresistant to a pathogen as compared to the wild type plant.(B) Genes encoding a Bacillus thuringiensis protein, a derivativethereof or a synthetic polypeptide modeled thereon. See, for example,Geiser, et al., (1986) Gene 48:109, who disclose the cloning andnucleotide sequence of a Bt delta-endotoxin gene. Moreover, DNAmolecules encoding delta-endotoxin genes can be purchased from AmericanType Culture Collection (Rockville, Md.), for example, under ATCC®Accession Numbers 40098, 67136, 31995 and 31998. Other non-limitingexamples of Bacillus thuringiensis transgenes being geneticallyengineered are given in the following patents and patent applicationsand hereby are incorporated by reference for this purpose: U.S. Pat.Nos. 5,188,960; 5,689,052; 5,880,275; 5,986,177; 6,023,013, 6,060,594,6,063,597, 6,077,824, 6,620,988, 6,642,030, 6,713,259, 6,893,826,7,105,332; 7,179,965, 7,208,474; 7,227,056, 7,288,643, 7,323,556,7,329,736, 7,449,552, 7,468,278, 7,510,878, 7,521,235, 7,544,862,7,605,304, 7,696,412, 7,629,504, 7,705,216, 7,772,465, 7,790,846,7,858,849 and WO 1991/14778; WO 1999/31248; WO 2001/12731; WO 1999/24581and WO 1997/40162.Genes encoding pesticidal proteins may also be stacked including but arenot limited to: insecticidal proteins from Pseudomonas sp. such asPSEEN3174 (Monalysin, (2011) PLoS Pathogens, 7:1-13), from Pseudomonasprotegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr,(2008) Environmental Microbiology 10:2368-2386: GenBank Accession No.EU400157); from Pseudomonas Taiwanensis (Liu, et al., (2010) J. Agric.Food Chem. 58:12343-12349) and from Pseudomonas pseudoalcligenes (Zhang,et al., (2009) Annals of Microbiology 59:45-50 and Li, et al., (2007)Plant Cell Tiss. Organ Cult. 89:159-168); insecticidal proteins fromPhotorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) TheOpen Toxinology Journal 3:101-118 and Morgan, et al., (2001) Applied andEnvir. Micro. 67:2062-2069), U.S. Pat. Nos. 6,048,838, and 6,379,946; aPIP-1 polypeptide of US Patent Publication US20140007292; an AfIP-1Aand/or AfIP-1B polypeptide of US Patent Publication US20140033361; aPHI-4 polypeptide of US patent Publication US20140274885 or PCT PatentPublication WO2014/150914; a PIP-47 polypeptide of PCT Serial NumberPCT/US14/51063, a PIP-72 polypeptide of PCT Serial NumberPCT/US14/55128, and δ-endotoxins including, but not limited to, theCry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11,Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21,Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry 28, Cry 29, Cry 30, Cry31,Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38, Cry39, Cry40, Cry41,Cry42, Cry43, Cry44, Cry45, Cry 46, Cry47, Cry49, Cry50, Cry51, Cry52,Cry53, Cry 54, Cry55, Cry56, Cry57, Cry58, Cry59, Cry60, Cry61, Cry62,Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70, Cry71, and Cry72 classes of δ-endotoxin genes and the B. thuringiensis cytolytic Cyt1and Cyt2 genes. Members of these classes of B. thuringiensisinsecticidal proteins include, but are not limited to Cry1Aa1 (Accession# AAA22353); Cry1Aa2 (Accession # Accession # AAA22552); Cry1Aa3(Accession # BAA00257); Cry1Aa4 (Accession # CAA31886); Cry1Aa5(Accession # BAA04468); Cry1Aa6 (Accession # AAA86265); Cry1Aa7(Accession # AAD46139); Cry1Aa8 (Accession # I26149); Cry1Aa9 (Accession# BAA77213); Cry1Aa10 (Accession # AAD55382); Cry1Aa11 (Accession #CAA70856); Cry1Aa12 (Accession # AAP80146); Cry1Aa13 (Accession #AAM44305); Cry1Aa14 (Accession # AAP40639); Cry1Aa15 (Accession #AAY66993); Cry1Aa16 (Accession # HQ439776); Cry1Aa17 (Accession #HQ439788); Cry1Aa18 (Accession # HQ439790); Cry1Aa19 (Accession #HQ685121); Cry1Aa20 (Accession # JF340156); Cry1Aa21 (Accession #JN651496); Cry1Aa22 (Accession # KC158223); Cry1Ab1 (Accession #AAA22330); Cry1Ab2 (Accession # AAA22613); Cry1Ab3 (Accession #AAA22561); Cry1Ab4 (Accession # BAA00071); Cry1Ab5 (Accession #CAA28405); Cry1Ab6 (Accession # AAA22420); Cry1Ab7 (Accession#CAA31620); Cry1Ab8 (Accession # AAA22551); Cry1Ab9 (Accession #CAA38701); Cry1Ab10 (Accession # A29125); Cry1Ab11 (Accession # I12419);Cry1Ab12 (Accession # AAC64003); Cry1Ab13 (Accession # AAN76494);Cry1Ab14 (Accession # AAG16877); Cry1Ab15 (Accession # AA013302);Cry1Ab16 (Accession # AAK55546); Cry1Ab17 (Accession # AAT46415);Cry1Ab18 (Accession # AAQ88259); Cry1Ab19 (Accession # AAW31761);Cry1Ab20 (Accession # ABB72460); Cry1Ab21 (Accession # ABS18384);Cry1Ab22 (Accession # ABW87320); Cry1Ab23 (Accession # HQ439777);Cry1Ab24 (Accession # HQ439778); Cry1Ab25 (Accession # HQ685122);Cry1Ab26 (Accession # HQ847729); Cry1Ab27 (Accession # JN135249);Cry1Ab28 (Accession # JN135250); Cry1Ab29 (Accession # JN135251);Cry1Ab30 (Accession # JN135252); Cry1Ab31 (Accession # JN135253);Cry1Ab32 (Accession # JN135254); Cry1Ab33 (Accession # AAS93798);Cry1Ab34 (Accession # KC156668); Cry1Ab-like (Accession # AAK14336);Cry1Ab-like (Accession # AAK14337); Cry1Ab-like (Accession # AAK14338);Cry1Ab-like (Accession # ABG88858); Cry1Ac1 (Accession # AAA22331);Cry1Ac2 (Accession # AAA22338); Cry1Ac3 (Accession # CAA38098); Cry1Ac4(Accession # AAA73077); Cry1Ac5 (Accession # AAA22339); Cry1Ac6(Accession # AAA86266); Cry1Ac7 (Accession # AAB46989); Cry1Ac8(Accession # AAC44841); Cry1Ac9 (Accession # AAB49768); Cry1Ac10(Accession # CAA05505); Cry1Ac11 (Accession # CAA10270); Cry1Ac12(Accession # I12418); Cry1Ac13 (Accession # AAD38701); Cry1Ac14(Accession # AAQ06607); Cry1Ac15 (Accession # AAN07788); Cry1Ac16(Accession # AAU87037); Cry1Ac17 (Accession # AAX18704); Cry1Ac18(Accession # AAY88347); Cry1Ac19 (Accession # ABD37053); Cry1Ac20(Accession # ABB89046); Cry1Ac21 (Accession # AAY66992); Cry1Ac22(Accession # ABZ01836); Cry1Ac23 (Accession # CAQ30431); Cry1Ac24(Accession # ABL01535); Cry1Ac25 (Accession # FJ513324); Cry1Ac26(Accession # FJ617446); Cry1Ac27 (Accession # FJ617447); Cry1Ac28(Accession # ACM90319); Cry1Ac29 (Accession # DQ438941); Cry1Ac30(Accession # GQ227507); Cry1Ac31 (Accession # GU446674); Cry1Ac32(Accession # HM061081); Cry1Ac33 (Accession # GQ866913); Cry1Ac34(Accession # HQ230364); Cry1Ac35 (Accession # JF340157); Cry1Ac36(Accession # JN387137); Cry1Ac37 (Accession # JQ317685); Cry1Ad1(Accession # AAA22340); Cry1Ad2 (Accession # CAA01880); Cry1Ae1(Accession # AAA22410); Cry1Af1 (Accession # AAB82749); Cry1Ag1(Accession # AAD46137); Cry1Ah1 (Accession # AAQ14326); Cry1Ah2(Accession # ABB76664); Cry1Ah3 (Accession # HQ439779); Cry1Ai1(Accession # AA039719); Cry1Ai2 (Accession # HQ439780); Cry1A-like(Accession # AAK14339); Cry1Ba1 (Accession # CAA29898); Cry1Ba2(Accession # CAA65003); Cry1Ba3 (Accession # AAK63251); Cry1Ba4(Accession # AAK51084); Cry1Ba5 (Accession # AB020894); Cry1Ba6(Accession # ABL60921); Cry1Ba7 (Accession # HQ439781); Cry1Bb1(Accession # AAA22344); Cry1Bb2 (Accession # HQ439782); Cry1Bc1(Accession # CAA86568); Cry1Bd1 (Accession # AAD10292); Cry1Bd2(Accession # AAM93496); Cry1Be1 (Accession # AAC32850); Cry1Be2(Accession # AAQ52387); Cry1Be3 (Accession # ACV96720); Cry1Be4(Accession # HM070026); Cry1Bf1 (Accession # CAC50778); Cry1Bf2(Accession # AAQ52380); Cry1Bg1 (Accession # AA039720); Cry1Bh1(Accession # HQ589331); Cry1Bi1 (Accession # KC156700); Cry1Ca1(Accession # CAA30396); Cry1Ca2 (Accession # CAA31951); Cry1Ca3(Accession # AAA22343); Cry1Ca4 (Accession # CAA01886); Cry1Ca5(Accession # CAA65457); Cry1Ca6 [1] (Accession # AAF37224); Cry1Ca7(Accession # AAG50438); Cry1Ca8 (Accession # AAM00264); Cry1Ca9(Accession # AAL79362); Cry1Ca10 (Accession # AAN16462); Cry1Ca11(Accession # AAX53094); Cry1Ca12 (Accession # HM070027); Cry1Ca13(Accession # HQ412621); Cry1Ca14 (Accession # JN651493); Cry1Cb1(Accession # M97880); Cry1Cb2 (Accession # AAG35409); Cry1Cb3 (Accession# ACD50894); Cry1Cb-like (Accession # AAX63901); Cry1Da1 (Accession #CAA38099); Cry1Da2 (Accession #176415); Cry1Da3 (Accession # HQ439784);Cry1Db1 (Accession # CAA80234); Cry1Db2 (Accession # AAK48937); Cry1Dc1(Accession # ABK35074); Cry1Ea1 (Accession # CAA37933); Cry1Ea2(Accession # CAA39609); Cry1Ea3 (Accession # AAA22345); Cry1Ea4(Accession # AADO4732); Cry1Ea5 (Accession # A15535); Cry1Ea6 (Accession# AAL50330); Cry1Ea7 (Accession # AAW72936); Cry1Ea8 (Accession #ABX11258); Cry1Ea9 (Accession # HQ439785); Cry1Ea10 (Accession #ADR00398); Cry1Ea11 (Accession # JQ652456); Cry1Eb1 (Accession #AAA22346); Cry1Fa1 (Accession # AAA22348); Cry1Fa2 (Accession #AAA22347); Cry1Fa3 (Accession # HM070028); Cry1Fa4 (Accession #HM439638); Cry1Fb1 (Accession # CAA80235); Cry1Fb2 (Accession #BAA25298); Cry1Fb3 (Accession # AAF21767); Cry1Fb4 (Accession #AAC10641); Cry1Fb5 (Accession # AAO13295); Cry1Fb6 (Accession #ACD50892); Cry1Fb7 (Accession # ACD50893); Cry1Ga1 (Accession #CAA80233); Cry1Ga2 (Accession # CAA70506); Cry1Gb1 (Accession #AAD10291); Cry1Gb2 (Accession # AA013756); Cry1Gc1 (Accession #AAQ52381); Cry1Ha1 (Accession # CAA80236); Cry1Hb1 (Accession #AAA79694); Cry1Hb2 (Accession # HQ439786); Cry1H-like (Accession #AAF01213); Cry1Ia1 (Accession # CAA44633); Cry1Ia2 (Accession #AAA22354); Cry1Ia3 (Accession # AAC36999); Cry1Ia4 (Accession #AAB00958); Cry1Ia5 (Accession # CAA70124); Cry1Ia6 (Accession #AAC26910); Cry1Ia7 (Accession # AAM73516); Cry1Ia8 (Accession #AAK66742); Cry1Ia9 (Accession # AAQ08616); Cry1Ia10 (Accession #AAP86782); Cry1Ia11 (Accession # CAC85964); Cry1Ia12 (Accession#AAV53390); Cry1Ia13 (Accession # ABF83202); Cry1Ia14 (Accession #ACG63871); Cry1Ia15 (Accession # FJ617445); Cry1Ia16 (Accession #FJ617448); Cry1Ia17 (Accession # GU989199); Cry1Ia18 (Accession #ADK23801); Cry1Ia19 (Accession # HQ439787); Cry1Ia20 (Accession #JQ228426); Cry1Ia21 (Accession # JQ228424); Cry1Ia22 (Accession #JQ228427); Cry1Ia23 (Accession # JQ228428); Cry1Ia24 (Accession #JQ228429); Cry1Ia25 (Accession # JQ228430); Cry1Ia26 (Accession #JQ228431); Cry1Ia27 (Accession # JQ228432); Cry1Ia28 (Accession #JQ228433); Cry1Ia29 (Accession # JQ228434); Cry1Ia30 (Accession #JQ317686); Cry1Ia31 (Accession # JX944038); Cry1Ia32 (Accession #JX944039); Cry1Ia33 (Accession # JX944040); Cry1Ib1 (Accession #AAA82114); Cry1Ib2 (Accession # ABW88019); Cry1Ib3 (Accession #ACD75515); Cry1Ib4 (Accession # HM051227); Cry1Ib5 (Accession #HM070028); Cry1Ib6 (Accession # ADK38579); Cry1Ib7 (Accession #JN571740); Cry1Ib8 (Accession # JN675714); Cry1Ib9 (Accession #JN675715); Cry1Ib10 (Accession # JN675716); Cry1Ib11 (Accession #JQ228423); Cry1Ic1 (Accession # AAC62933); Cry1Ic2 (Accession #AAE71691); Cry1Id1 (Accession # AAD44366); Cry1Id2 (Accession #JQ228422); Cry1Ie1 (Accession # AAG43526); Cry1Ie2 (Accession #HM439636); Cry1Ie3 (Accession # KC156647); Cry1Ie4 (Accession #KC156681); Cry1If1 (Accession # AAQ52382); Cry1Ig1 (Accession #KC156701); Cry1I-like (Accession # AAC31094); Cry1I-like (Accession #ABG88859); Cry1Ja1 (Accession # AAA22341); Cry1Ja2 (Accession #HM070030); Cry1Ja3 (Accession # JQ228425); Cry1Jb1 (Accession #AAA98959); Cry1Jc1 (Accession # AAC31092); Cry1Jc2 (Accession #AAQ52372); Cry1Jd1 (Accession # CAC50779); Cry1Ka1 (Accession #AAB00376); Cry1Ka2 (Accession # HQ439783); Cry1La1 (Accession #AAS60191); Cry1La2 (Accession # HM070031); Cry1Ma1 (Accession #FJ884067); Cry1Ma2 (Accession # KC156659); Cry1Na1 (Accession #KC156648); Cry1Nb1 (Accession # KC156678); Cry1-like (Accession #AAC31091); Cry2Aa1 (Accession # AAA22335); Cry2Aa2 (Accession #AAA83516); Cry2Aa3 (Accession # D86064); Cry2Aa4 (Accession # AAC04867);Cry2Aa5 (Accession # CAA10671); Cry2Aa6 (Accession # CAA10672); Cry2Aa7(Accession # CAA10670); Cry2Aa8 (Accession # AA013734); Cry2Aa9(Accession # AA013750); Cry2Aa10 (Accession # AAQ04263); Cry2Aa11(Accession # AAQ52384); Cry2Aa12 (Accession # AB183671); Cry2Aa13(Accession # ABL01536); Cry2Aa14 (Accession # ACF04939); Cry2Aa15(Accession # JN426947); Cry2Ab1 (Accession # AAA22342); Cry2Ab2(Accession # CAA39075); Cry2Ab3 (Accession # AAG36762); Cry2Ab4(Accession # AAO13296); Cry2Ab5 (Accession # AAQ04609); Cry2Ab6(Accession # AAP59457); Cry2Ab7 (Accession # AAZ66347); Cry2Ab8(Accession # ABC95996); Cry2Ab9 (Accession # ABC74968); Cry2Ab10(Accession # EF157306); Cry2Ab11 (Accession # CAM84575); Cry2Ab12(Accession # ABM21764); Cry2Ab13 (Accession # ACG76120); Cry2Ab14(Accession # ACG76121); Cry2Ab15 (Accession # HM037126); Cry2Ab16(Accession # GQ866914); Cry2Ab17 (Accession # HQ439789); Cry2Ab18(Accession # JN135255); Cry2Ab19 (Accession # JN135256); Cry2Ab20(Accession # JN135257); Cry2Ab21 (Accession # JN135258); Cry2Ab22(Accession # JN135259); Cry2Ab23 (Accession # JN135260); Cry2Ab24(Accession # JN135261); Cry2Ab25 (Accession # JN415485); Cry2Ab26(Accession # JN426946); Cry2Ab27 (Accession # JN415764); Cry2Ab28(Accession # JN651494); Cry2Ac1 (Accession # CAA40536); Cry2Ac2(Accession # AAG35410); Cry2Ac3 (Accession # AAQ52385); Cry2Ac4(Accession # ABC95997); Cry2Ac5 (Accession # ABC74969); Cry2Ac6(Accession # ABC74793); Cry2Ac7 (Accession # CAL18690); Cry2Ac8(Accession # CAM09325); Cry2Ac9 (Accession # CAM09326); Cry2Ac10(Accession # ABN15104); Cry2Ac11 (Accession # CAM83895); Cry2Ac12(Accession # CAM83896); Cry2Ad1 (Accession # AAF09583); Cry2Ad2(Accession # ABC86927); Cry2Ad3 (Accession # CAK29504); Cry2Ad4(Accession # CAM32331); Cry2Ad5 (Accession # CA078739); Cry2Ae1(Accession # AAQ52362); Cry2Af1 (Accession # AB030519); Cry2Af2(Accession # GQ866915); Cry2Ag1 (Accession # ACH91610); Cry2Ah1(Accession # EU939453); Cry2Ah2 (Accession # ACL80665); Cry2Ah3(Accession # GU073380); Cry2Ah4 (Accession # KC156702); Cry2Ai1(Accession # FJ788388); Cry2Aj (Accession #); Cry2Ak1 (Accession #KC156660); Cry2Ba1 (Accession # KC156658); Cry3Aa1 (Accession #AAA22336); Cry3Aa2 (Accession # AAA22541); Cry3Aa3 (Accession #CAA68482); Cry3Aa4 (Accession # AAA22542); Cry3Aa5 (Accession #AAA50255); Cry3Aa6 (Accession # AAC43266); Cry3Aa7 (Accession #CAB41411); Cry3Aa8 (Accession # AAS79487); Cry3Aa9 (Accession #AAW05659); Cry3Aa10 (Accession # AAU29411); Cry3Aa11 (Accession #AAW82872); Cry3Aa12 (Accession # ABY49136); Cry3Ba1 (Accession #CAA34983); Cry3Ba2 (Accession # CAA00645); Cry3Ba3 (Accession #JQ397327); Cry3Bb1 (Accession # AAA22334); Cry3Bb2 (Accession #AAA74198); Cry3Bb3 (Accession # I15475); Cry3Ca1 (Accession # CAA42469);Cry4Aa1 (Accession # CAA68485); Cry4Aa2 (Accession # BAA00179); Cry4Aa3(Accession # CAD30148); Cry4Aa4 (Accession # AFB18317); Cry4A-like(Accession # AAY96321); Cry4Ba1 (Accession # CAA30312); Cry4Ba2(Accession # CAA30114); Cry4Ba3 (Accession # AAA22337); Cry4Ba4(Accession # BAA00178); Cry4Ba5 (Accession # CAD30095); Cry4Ba-like(Accession # ABC47686); Cry4Ca1 (Accession # EU646202); Cry4Cb1(Accession # FJ403208); Cry4Cb2 (Accession # FJ597622); Cry4Cc1(Accession # FJ403207); Cry5Aa1 (Accession # AAA67694); Cry5Ab1(Accession # AAA67693); Cry5Ac1 (Accession # I34543); Cry5Ad1 (Accession# ABQ82087); Cry5Ba1 (Accession # AAA68598); Cry5Ba2 (Accession #ABW88931); Cry5Ba3 (Accession # AFJ04417); Cry5Ca1 (Accession #HM461869); Cry5Ca2 (Accession # ZP_04123426); Cry5Da1 (Accession #HM461870); Cry5Da2 (Accession # ZP_04123980); Cry5Ea1 (Accession #HM485580); Cry5Ea2 (Accession # ZP_04124038); Cry6Aa1 (Accession #AAA22357); Cry6Aa2 (Accession # AAM46849); Cry6Aa3 (Accession #ABH03377); Cry6Ba1 (Accession # AAA22358); Cry7Aa1 (Accession #AAA22351); Cry7Ab1 (Accession # AAA21120); Cry7Ab2 (Accession #AAA21121); Cry7Ab3 (Accession # ABX24522); Cry7Ab4 (Accession #EU380678); Cry7Ab5 (Accession # ABX79555); Cry7Ab6 (Accession #AC144005); Cry7Ab7 (Accession # ADB89216); Cry7Ab8 (Accession #GU145299); Cry7Ab9 (Accession # ADD92572); Cry7Ba1 (Accession #ABB70817); Cry7Bb1 (Accession # KC156653); Cry7Ca1 (Accession #ABR67863); Cry7Cb1 (Accession # KC156698); Cry7Da1 (Accession #ACQ99547); Cry7Da2 (Accession # HM572236); Cry7Da3 (Accession #KC156679); Cry7Ea1 (Accession # HM035086); Cry7Ea2 (Accession #HM132124); Cry7Ea3 (Accession # EEM19403); Cry7Fa1 (Accession #HM035088); Cry7Fa2 (Accession # EEM19090); Cry7Fb1 (Accession #HM572235); Cry7Fb2 (Accession # KC156682); Cry7Ga1 (Accession #HM572237); Cry7Ga2 (Accession # KC156669); Cry7Gb1 (Accession #KC156650); Cry7Gc1 (Accession # KC156654); Cry7Gd1 (Accession #KC156697); Cry7Ha1 (Accession # KC156651); Cry7Ia1 (Accession #KC156665); Cry7Ja1 (Accession # KC156671); Cry7Ka1 (Accession #KC156680); Cry7Kb1 (Accession # BAM99306); Cry7La1 (Accession #BAM99307); Cry8Aa1 (Accession # AAA21117); Cry8Ab1 (Accession #EU044830); Cry8Ac1 (Accession # KC156662); Cry8Ad1 (Accession #KC156684); Cry8Ba1 (Accession # AAA21118); Cry8Bb1 (Accession #CAD57542); Cry8Bc1 (Accession # CAD57543); Cry8Ca1 (Accession #AAA21119); Cry8Ca2 (Accession # AAR98783); Cry8Ca3 (Accession #EU625349); Cry8Ca4 (Accession # ADB54826); Cry8Da1 (Accession #BAC07226); Cry8Da2 (Accession # BD133574); Cry8Da3 (Accession #BD133575); Cry8Db1 (Accession # BAF93483); Cry8Ea1 (Accession #AAQ73470); Cry8Ea2 (Accession # EU047597); Cry8Ea3 (Accession #KC855216); Cry8Fa1 (Accession # AAT48690); Cry8Fa2 (Accession #HQ174208); Cry8Fa3 (Accession # AFH78109); Cry8Ga1 (Accession #AAT46073); Cry8Ga2 (Accession # ABC42043); Cry8Ga3 (Accession #FJ198072); Cry8Ha1 (Accession # AAW81032); Cry8Ia1 (Accession #EU381044); Cry8Ia2 (Accession # GU073381); Cry8Ia3 (Accession #HM044664); Cry8Ia4 (Accession # KC156674); Cry8Ib1 (Accession #GU325772); Cry8Ib2 (Accession # KC156677); Cry8Ja1 (Accession #EU625348); Cry8Ka1 (Accession # FJ422558); Cry8Ka2 (Accession #ACN87262); Cry8Kb1 (Accession # HM123758); Cry8Kb2 (Accession #KC156675); Cry8La1 (Accession #GU325771); Cry8Ma1 (Accession #HM044665); Cry8Ma2 (Accession # EEM86551); Cry8Ma3 (Accession #HM210574); Cry8Na1 (Accession # HM640939); Cry8Pa1 (Accession #HQ388415); Cry8Qa1 (Accession # HQ441166); Cry8Qa2 (Accession #KC152468); Cry8Ra1 (Accession # AFP87548); Cry8Sa1 (Accession #JQ740599); Cry8Ta1 (Accession # KC156673); Cry8-like (Accession #FJ770571); Cry8-like (Accession # ABS53003); Cry9Aa1 (Accession #CAA41122); Cry9Aa2 (Accession # CAA41425); Cry9Aa3 (Accession #GQ249293); Cry9Aa4 (Accession # GQ249294); Cry9Aa5 (Accession #JX174110); Cry9Aa like (Accession # AAQ52376); Cry9Ba1 (Accession #CAA52927); Cry9Ba2 (Accession # GU299522); Cry9Bb1 (Accession #AAV28716); Cry9Ca1 (Accession # CAA85764); Cry9Ca2 (Accession #AAQ52375); Cry9Da1 (Accession # BAA19948); Cry9Da2 (Accession #AAB97923); Cry9Da3 (Accession # GQ249293); Cry9Da4 (Accession #GQ249297); Cry9Db1 (Accession # AAX78439); Cry9Dc1 (Accession #KC156683); Cry9Ea1 (Accession # BAA34908); Cry9Ea2 (Accession #AAO12908); Cry9Ea3 (Accession # ABM21765); Cry9Ea4 (Accession #ACE88267); Cry9Ea5 (Accession # ACF04743); Cry9Ea6 (Accession #ACG63872); Cry9Ea7 (Accession # FJ380927); Cry9Ea8 (Accession #GQ249292); Cry9Ea9 (Accession # JN651495); Cry9Eb1 (Accession #CAC50780); Cry9Eb2 (Accession # GQ249298); Cry9Eb3 (Accession #KC156646); Cry9Ec1 (Accession # AAC63366); Cry9Ed1 (Accession #AAX78440); Cry9Ee1 (Accession # GQ249296); Cry9Ee2 (Accession #KC156664); Cry9Fa1 (Accession # KC156692); Cry9Ga1 (Accession #KC156699); Cry9-like (Accession # AAC63366); Cry10Aa1 (Accession #AAA22614); Cry10Aa2 (Accession # E00614); Cry10Aa3 (Accession #CAD30098); Cry10Aa4 (Accession # AFB18318); Cry10A-like (Accession #DQ167578); Cry11Aa1 (Accession # AAA22352); Cry11Aa2 (Accession #AAA22611); Cry11Aa3 (Accession # CAD30081); Cry11Aa4 (Accession #AFB18319); Cry1Aa-like (Accession # DQ166531); Cry11Ba1 (Accession #CAA60504); Cry11Bb1 (Accession # AAC97162); Cry11Bb2 (Accession #HM068615); Cry12Aa1 (Accession # AAA22355); Cry13Aa1 (Accession #AAA22356); Cry14Aa1 (Accession # AAA21516); Cry14Ab1 (Accession #KC156652); Cry15Aa1 (Accession # AAA22333); Cry16Aa1 (Accession #CAA63860); Cry17Aa1 (Accession # CAA67841); Cry18Aa1 (Accession #CAA67506); Cry18Ba1 (Accession # AAF89667); Cry18Ca1 (Accession #AAF89668); Cry19Aa1 (Accession # CAA68875); Cry19Ba1 (Accession #BAA32397); Cry19Ca1 (Accession # AFM37572); Cry20Aa1 (Accession #AAB93476); Cry20Ba1 (Accession # ACS93601); Cry20Ba2 (Accession #KC156694); Cry20-like (Accession # GQ144333); Cry21Aa1 (Accession #I32932); Cry21Aa2 (Accession # I66477); Cry21Ba1 (Accession # BAC06484);Cry21Ca1 (Accession # JF521577); Cry21Ca2 (Accession # KC156687);Cry21Da1 (Accession # JF521578); Cry22Aa1 (Accession # I34547); Cry22Aa2(Accession # CAD43579); Cry22Aa3 (Accession # ACD93211); Cry22Ab1(Accession # AAK50456); Cry22Ab2 (Accession # CAD43577); Cry22Ba1(Accession # CAD43578); Cry22Bb1 (Accession # KC156672); Cry23Aa1(Accession # AAF76375); Cry24Aa1 (Accession # AAC61891); Cry24Ba1(Accession # BAD32657); Cry24Ca1 (Accession # CAJ43600); Cry25Aa1(Accession # AAC61892); Cry26Aa1 (Accession # AAD25075); Cry27Aa1(Accession # BAA82796); Cry28Aa1 (Accession # AAD24189); Cry28Aa2(Accession # AAG00235); Cry29Aa1 (Accession # CAC80985); Cry30Aa1(Accession # CAC80986); Cry30Ba1 (Accession # BADO0052); Cry30Ca1(Accession # BAD67157); Cry30Ca2 (Accession # ACU24781); Cry30Da1(Accession # EF095955); Cry30Db1 (Accession # BAE80088); Cry30Ea1(Accession # ACC95445); Cry30Ea2 (Accession # FJ499389); Cry30Fa1(Accession # AC122625); Cry30Ga1 (Accession # ACG60020); Cry30Ga2(Accession # HQ638217); Cry31Aa1 (Accession # BAB11757); Cry31Aa2(Accession # AAL87458); Cry31Aa3 (Accession # BAE79808); Cry31Aa4(Accession # BAF32571); Cry31 Aa5 (Accession # BAF32572); Cry31Aa6(Accession # BA144026); Cry31Ab1 (Accession # BAE79809); Cry31Ab2(Accession # BAF32570); Cry31Ac1 (Accession # BAF34368); Cry31Ac2(Accession # AB731600); Cry31Ad1 (Accession # BA144022); Cry32Aa1(Accession # AAG36711); Cry32Aa2 (Accession # GU063849); Cry32Ab1(Accession # GU063850); Cry32Ba1 (Accession # BAB78601); Cry32Ca1(Accession # BAB78602); Cry32Cb1 (Accession # KC156708); Cry32Da1(Accession # BAB78603); Cry32Ea1 (Accession # GU324274); Cry32Ea2(Accession # KC156686); Cry32Eb1 (Accession # KC156663); Cry32Fa1(Accession # KC156656); Cry32Ga1 (Accession # KC156657); Cry32Ha1(Accession # KC156661); Cry32Hb1 (Accession # KC156666); Cry32Ia1(Accession # KC156667); Cry32Ja1 (Accession # KC156685); Cry32Ka1(Accession # KC156688); Cry32La1 (Accession # KC156689); Cry32Ma1(Accession # KC156690); Cry32Mb1 (Accession # KC156704); Cry32Na1(Accession # KC156691); Cry32Oa1 (Accession # KC156703); Cry32Pa1(Accession # KC156705); Cry32Qa1 (Accession # KC156706); Cry32Ra1(Accession # KC156707); Cry32Sa1 (Accession # KC156709); Cry32Ta1(Accession # KC156710); Cry32Ua1 (Accession # KC156655); Cry33Aa1(Accession # AAL26871); Cry34Aa1 (Accession # AAG50341); Cry34Aa2(Accession # AAK64560); Cry34Aa3 (Accession # AAT29032); Cry34Aa4(Accession # AAT29030); Cry34Ab1 (Accession # AAG41671); Cry34Ac1(Accession # AAG50118); Cry34Ac2 (Accession # AAK64562); Cry34Ac3(Accession # AAT29029); Cry34Ba1 (Accession # AAK64565); Cry34Ba2(Accession # AAT29033); Cry34Ba3 (Accession # AAT29031); Cry35Aa1(Accession # AAG50342); Cry35Aa2 (Accession # AAK64561); Cry35Aa3(Accession # AAT29028); Cry35Aa4 (Accession # AAT29025); Cry35Ab1(Accession # AAG41672); Cry35Ab2 (Accession # AAK64563); Cry35Ab3(Accession # AY536891); Cry35Ac1 (Accession # AAG50117); Cry35Ba1(Accession # AAK64566); Cry35Ba2 (Accession # AAT29027); Cry35Ba3(Accession # AAT29026); Cry36Aa1 (Accession # AAK64558); Cry37Aa1(Accession # AAF76376); Cry38Aa1 (Accession # AAK64559); Cry39Aa1(Accession # BAB72016); Cry40Aa1 (Accession # BAB72018); Cry40Ba1(Accession # BAC77648); Cry40Ca1 (Accession # EU381045); Cry40Da1(Accession # ACF15199); Cry41Aa1 (Accession # BAD35157); Cry41Ab1(Accession # BAD35163); Cry41Ba1 (Accession # HM461871); Cry41Ba2(Accession # ZP_04099652); Cry42Aa1 (Accession # BAD35166); Cry43Aa1(Accession # BAD15301); Cry43Aa2 (Accession # BAD95474); Cry43Ba1(Accession # BAD15303); Cry43Ca1 (Accession # KC156676); Cry43Cb1(Accession # KC156695); Cry43Cc1 (Accession # KC156696); Cry43-like(Accession # BAD15305); Cry44Aa (Accession # BADO8532); Cry45Aa(Accession # BAD22577); Cry46Aa (Accession # BAC79010); Cry46Aa2(Accession # BAG68906); Cry46Ab (Accession # BAD35170); Cry47Aa(Accession # AAY24695); Cry48Aa (Accession # CAJ18351); Cry48Aa2(Accession # CAJ86545); Cry48Aa3 (Accession # CAJ86546); Cry48Ab(Accession # CAJ86548); Cry48Ab2 (Accession # CAJ86549); Cry49Aa(Accession # CAH56541); Cry49Aa2 (Accession # CAJ86541); Cry49Aa3(Accession # CAJ86543); Cry49Aa4 (Accession # CAJ86544); Cry49Ab1(Accession # CAJ86542); Cry50Aa1 (Accession # BAE86999); Cry50Ba1(Accession # GU446675); Cry50Ba2 (Accession # GU446676); Cry51Aa1(Accession # AB114444); Cry51Aa2 (Accession # GU570697); Cry52Aa1(Accession # EF613489); Cry52Ba1 (Accession # FJ361760); Cry53Aa1(Accession # EF633476); Cry53Ab1 (Accession # FJ361759); Cry54Aa1(Accession # ACA52194); Cry54Aa2 (Accession # GQ140349); Cry54Ba1(Accession # GU446677); Cry55Aa1 (Accession # ABW88932); Cry54Ab1(Accession # JQ916908); Cry55Aa2 (Accession # AAE33526); Cry56Aa1(Accession # ACU57499); Cry56Aa2 (Accession # GQ483512); Cry56Aa3(Accession # JX025567); Cry57Aa1 (Accession # ANC87261); Cry58Aa1(Accession # ANC87260); Cry59Ba1 (Accession # JN790647); Cry59Aa1(Accession # ACR43758); Cry60Aa1 (Accession # ACU24782); Cry60Aa2(Accession # EA057254); Cry60Aa3 (Accession # EEM99278); Cry60Ba1(Accession # GU810818); Cry60Ba2 (Accession # EA057253); Cry60Ba3(Accession # EEM99279); Cry61Aa1 (Accession # HM035087); Cry61Aa2(Accession # HM132125); Cry61Aa3 (Accession # EEM19308); Cry62Aa1(Accession # HM054509); Cry63Aa1 (Accession # BA144028); Cry64Aa1(Accession # BAJ05397); Cry65Aa1 (Accession # HM461868); Cry65Aa2(Accession # ZP_04123838); Cry66Aa1 (Accession # HM485581); Cry66Aa2(Accession # ZP_04099945); Cry67Aa1 (Accession # HM485582); Cry67Aa2(Accession # ZP_04148882); Cry68Aa1 (Accession # HQ113114); Cry69Aa1(Accession # HQ401006); Cry69Aa2 (Accession # JQ821388); Cry69Ab1(Accession # JN209957); Cry70Aa1 (Accession # JN646781); Cry70Ba1(Accession # ADO51070); Cry70Bb1 (Accession # EEL67276); Cry71Aa1(Accession # JX025568); Cry72Aa1 (Accession # JX025569).

Examples of δ-endotoxins also include but are not limited to Cry1Aproteins of U.S. Pat. Nos. 5,880,275 and 7,858,849; a DIG-3 or DIG-11toxin (N-terminal deletion of α-helix 1 and/or α-helix 2 variants of Cryproteins such as Cry1A) of U.S. Pat. Nos. 8,304,604 and 8,304,605, Cry1Bof U.S. patent application Ser. No. 10/525,318; Cry1C of U.S. Pat. No.6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960, 6,218,188; Cry1A/Fchimeras of U.S. Pat. Nos. 7,070,982; 6,962,705 and 6,713,063); a Cry2protein such as Cry2Ab protein of U.S. Pat. No. 7,064,249); a Cry3Aprotein including but not limited to an engineered hybrid insecticidalprotein (eHIP) created by fusing unique combinations of variable regionsand conserved blocks of at least two different Cry proteins (US PatentApplication Publication Number 2010/0017914); a Cry4 protein; a Cry5protein; a Cry6 protein; Cry8 proteins of U.S. Pat. Nos. 7,329,736,7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378,499 and 7,462,760; aCry9 protein such as such as members of the Cry9A, Cry9B, Cry9C, Cry9D,Cry9E, and Cry9F families; a Cry15 protein of Naimov, et al., (2008)Applied and Environmental Microbiology 74:7145-7151; a Cry22, a Cry34Ab1protein of U.S. Pat. Nos. 6,127,180, 6,624,145 and 6,340,593; a CryET33and CryET34 protein of U.S. Pat. Nos. 6,248,535, 6,326,351, 6,399,330,6,949,626, 7,385,107 and 7,504,229; a CryET33 and CryET34 homologs of USPatent Publication Number 2006/0191034, 2012/0278954, and PCTPublication Number WO 2012/139004; a Cry35Ab1 protein of U.S. Pat. Nos.6,083,499, 6,548,291 and 6,340,593; a Cry46 protein, a Cry 51 protein, aCry binary toxin; a TIC901 or related toxin; TIC807 of US 2008/0295207;ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US2006/033867; AXMI-027, AXMI-036, and AXMI-038 of U.S. Pat. No.8,236,757; AXMI-031, AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. No.7,923,602; AXMI-018, AXMI-020, and AXMI-021 of WO 2006/083891; AXMI-010of WO 2005/038032; AXMI-003 of WO 2005/021585; AXMI-008 of US2004/0250311; AXMI-006 of US 2004/0216186; AXMI-007 of US 2004/0210965;AXMI-009 of US 2004/0210964; AXMI-014 of US 2004/0197917; AXMI-004 of US2004/0197916; AXMI-028 and AXMI-029 of WO 2006/119457; AXMI-007,AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO2004/074462; AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 ofUS20110023184; AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019,AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023,AXMI-041, AXMI-063, and AXMI-064 of US 2011/0263488; AXMI-R1 and relatedproteins of US 2010/0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z andAXMI225z of WO 2011/103248; AXMI218, AXMI219, AXMI220, AXM1226, AXM1227,AXM1228, AXM1229, AXMI230, and AXMI231 of WO11/103247; AXMI-115,AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. 8,334,431;AXMI-001, AXMI-002, AXMI-030, AXMI-035, and AXMI-045 of US 2010/0298211;AXMI-066 and AXMI-076 of US20090144852; AXMI128, AXMI130, AXMI131,AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148,AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158,AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171,AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179,AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189of U.S. Pat. No. 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082, AXMI091,AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102,AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, AXMI112,AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122,AXMI123, AXMI124, AXM11257, AXM11268, AXMI127, AXMI129, AXMI164,AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of US 2010/0005543;and Cry proteins such as Cry1A and Cry3A having modified proteolyticsites of U.S. Pat. No. 8,319,019; and a Cry1Ac, Cry2Aa and Cry1Ca toxinprotein from Bacillus thuringiensis strain VBTS 2528 of US PatentApplication Publication Number 2011/0064710. Other Cry proteins are wellknown to one skilled in the art (see, Crickmore, et al., “Bacillusthuringiensis toxin nomenclature” (2011), atlifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ which can be accessed onthe world-wide web using the “www” prefix). The insecticidal activity ofCry proteins is well known to one skilled in the art (for review, see,van Frannkenhuyzen, (2009) J. Invert. Path. 101:1-16). The use of Cryproteins as transgenic plant traits is well known to one skilled in theart and Cry-transgenic plants including but not limited to Cry1Ac,Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab,Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c andCBI-Bt have received regulatory approval (see, Sanahuja, (2011) PlantBiotech Journal 9:283-300 and the CERA (2010) GM Crop Database Centerfor Environmental Risk Assessment (CERA), ILSI Research Foundation,Washington D.C. at cera-gmc.org/index.php?action=gm_crop_database whichcan be accessed on the world-wide web using the “www” prefix). More thanone pesticidal proteins well known to one skilled in the art can also beexpressed in plants such as Vip3Ab & Cry1Fa (US2012/0317682), Cry1BE &Cry1F (US2012/0311746), Cry1CA & Cry1AB (US2012/0311745), Cry1F & CryCa(US2012/0317681), Cry1DA & Cry1BE (US2012/0331590), Cry1DA & Cry1Fa(US2012/0331589), Cry1AB & Cry1BE (US2012/0324606), and Cry1Fa & Cry2Aa,Cry1I or Cry1E (US2012/0324605). Pesticidal proteins also includeinsecticidal lipases including lipid acyl hydrolases of U.S. Pat. No.7,491,869, and cholesterol oxidases such as from Streptomyces (Purcellet al. (1993) Biochem Biophys Res Commun 15:1406-1413). Pesticidalproteins also include VIP (vegetative insecticidal proteins) toxins ofU.S. Pat. Nos. 5,877,012, 6,107,279, 6,137,033, 7,244,820, 7,615,686,and 8,237,020, and the like. Other VIP proteins are well known to oneskilled in the art (see,lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html which can beaccessed on the world-wide web using the “www” prefix). Pesticidalproteins also include toxin complex (TC) proteins, obtainable fromorganisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, U.S.Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have “stand alone”insecticidal activity and other TC proteins enhance the activity of thestand-alone toxins produced by the same given organism. The toxicity ofa “stand-alone” TC protein (from Photorhabdus, Xenorhabdus orPaenibacillus, for example) can be enhanced by one or more TC protein“potentiators” derived from a source organism of a different genus.There are three main types of TC proteins. As referred to herein, ClassA proteins (“Protein A”) are stand-alone toxins. Class B proteins(“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity ofClass A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 andXptA2. Examples of Class B proteins are TcaC, TcdB, XptB1Xb and XptC1Wi.Examples of Class C proteins are TccC, XptC1Xb and XptB1Wi. Pesticidalproteins also include spider, snake and scorpion venom proteins.Examples of spider venom peptides include but are not limited tolycotoxin-1 peptides and mutants thereof (U.S. Pat. No. 8,334,366).

(C) A polynucleotide encoding an insect-specific hormone or pheromonesuch as an ecdysteroid and juvenile hormone, a variant thereof, amimetic based thereon or an antagonist or agonist thereof. See, forexample, the disclosure by Hammock, et al., (1990) Nature 344:458, ofbaculovirus expression of cloned juvenile hormone esterase, aninactivator of juvenile hormone.(D) A polynucleotide encoding an insect-specific peptide which, uponexpression, disrupts the physiology of the affected pest. For example,see the disclosures of, Regan, (1994) J. Biol. Chem. 269:9 (expressioncloning yields DNA coding for insect diuretic hormone receptor); Pratt,et al., (1989) Biochem. Biophys. Res. Comm. 163:1243 (an allostatin isidentified in Diploptera puntata); Chattopadhyay, et al., (2004)Critical Reviews in Microbiology 30(1):33-54; Zjawiony, (2004) J NatProd 67(2):300-310; Carlini and Grossi-de-Sa, (2002) Toxicon40(11):1515-1539; Ussuf, et al., (2001) Curr Sci. 80(7):847-853 andVasconcelos and Oliveira, (2004) Toxicon 44(4):385-403. See also, U.S.Pat. No. 5,266,317 to Tomalski, et al., who disclose genes encodinginsect-specific toxins.(E) A polynucleotide encoding an enzyme responsible for ahyperaccumulation of a monoterpene, a sesquiterpene, a steroid,hydroxamic acid, a phenylpropanoid derivative or another non-proteinmolecule with insecticidal activity.(F) A polynucleotide encoding an enzyme involved in the modification,including the post-translational modification, of a biologically activemolecule; for example, a glycolytic enzyme, a proteolytic enzyme, alipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, ahydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, anelastase, a chitinase and a glucanase, whether natural or synthetic.See, PCT Application WO 1993/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase gene. DNA molecules whichcontain chitinase-encoding sequences can be obtained, for example, fromthe ATCC® under Accession Numbers 39637 and 67152. See also, Kramer, etal., (1993) Insect Biochem. Molec. Biol. 23:691, who teach thenucleotide sequence of a cDNA encoding tobacco hookworm chitinase andKawalleck, et al., (1993) Plant Molec. Biol. 21:673, who provide thenucleotide sequence of the parsley ubi4-2 polyubiquitin gene, and U.S.Pat. Nos. 6,563,020; 7,145,060 and 7,087,810.(G) A polynucleotide encoding a molecule that stimulates signaltransduction. For example, see the disclosure by Botella, et al., (1994)Plant Molec. Biol. 24:757, of nucleotide sequences for mung beancalmodulin cDNA clones, and Griess, et al., (1994) Plant Physiol.104:1467, who provide the nucleotide sequence of a maize calmodulin cDNAclone.(H) A polynucleotide encoding a hydrophobic moment peptide. See, PCTApplication WO 1995/16776 and U.S. Pat. No. 5,580,852 disclosure ofpeptide derivatives of Tachyplesin which inhibit fungal plant pathogens)and PCT Application WO 1995/18855 and U.S. Pat. No. 5,607,914 (teachessynthetic antimicrobial peptides that confer disease resistance).(I) A polynucleotide encoding a membrane permease, a channel former or achannel blocker. For example, see the disclosure by Jaynes, et al.,(1993) Plant Sci. 89:43, of heterologous expression of a cecropin-betalytic peptide analog to render transgenic tobacco plants resistant toPseudomonas solanacearum.(J) A gene encoding a viral-invasive protein or a complex toxin derivedtherefrom. For example, the accumulation of viral coat proteins intransformed plant cells imparts resistance to viral infection and/ordisease development effected by the virus from which the coat proteingene is derived, as well as by related viruses. See, Beachy, et al.,(1990) Ann. Rev. Phytopathol. 28:451. Coat protein-mediated resistancehas been conferred upon transformed plants against alfalfa mosaic virus,cucumber mosaic virus, tobacco streak virus, potato virus X, potatovirus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaicvirus. Id.(K) A gene encoding an insect-specific antibody or an immunotoxinderived therefrom. Thus, an antibody targeted to a critical metabolicfunction in the insect gut would inactivate an affected enzyme, killingthe insect. Cf. Taylor, et al., Abstract #497, SEVENTH INT'L SYMPOSIUMON MOLECULAR PLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994)(enzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments).(L) A gene encoding a virus-specific antibody. See, for example,Tavladoraki, et al., (1993) Nature 366:469, who show that transgenicplants expressing recombinant antibody genes are protected from virusattack.(M) A polynucleotide encoding a developmental-arrestive protein producedin nature by a pathogen or a parasite. Thus, fungal endoalpha-1,4-D-polygalacturonases facilitate fungal colonization and plantnutrient release by solubilizing plant cell wallhomo-alpha-1,4-D-galacturonase. See, Lamb, et al., (1992) Bio/Technology10:1436. The cloning and characterization of a gene which encodes a beanendopolygalacturonase-inhibiting protein is described by Toubart, etal., (1992) Plant J. 2:367.(N) A polynucleotide encoding a developmental-arrestive protein producedin nature by a plant. For example, Logemann, et al., (1992)Bio/Technology 10:305, have shown that transgenic plants expressing thebarley ribosome-inactivating gene have an increased resistance to fungaldisease.(O) Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes. Briggs, (1995) Current Biology5(2), Pieterse and Van Loon, (2004) Curr. Opin. Plant Bio. 7(4):456-64and Somssich, (2003) Cell 113(7):815-6.(P) Antifungal genes (Cornelissen and Melchers, (1993) Pl. Physiol.101:709-712 and Parijs, et al., (1991) Planta 183:258-264 and Bushnell,et al., (1998) Can. J. of Plant Path. 20(2):137-149. Also see, U.S.patent application Ser. Nos. 09/950,933; 11/619,645; 11/657,710;11/748,994; 11/774,121 and U.S. Pat. Nos. 6,891,085 and 7,306,946. LysMReceptor-like kinases for the perception of chitin fragments as a firststep in plant defense response against fungal pathogens (US2012/0110696).(Q) Detoxification genes, such as for fumonisin, beauvericin,moniliformin and zearalenone and their structurally related derivatives.For example, see, U.S. Pat. Nos. 5,716,820; 5,792,931; 5,798,255;5,846,812; 6,083,736; 6,538,177; 6,388,171 and 6,812,380.(R) A polynucleotide encoding a Cystatin and cysteine proteinaseinhibitors. See, U.S. Pat. No. 7,205,453.(S) Defensin genes. See, WO 2003/000863 and U.S. Pat. Nos. 6,911,577;6,855,865; 6,777,592 and 7,238,781.(T) Genes conferring resistance to nematodes. See, e.g., PCT ApplicationWO 1996/30517; PCT Application WO 1993/19181, WO 2003/033651 and Urwin,et al., (1998) Planta 204:472-479, Williamson, (1999) Curr Opin PlantBio. 2(4):327-31; U.S. Pat. Nos. 6,284,948 and 7,301,069 and miR164genes (WO 2012/058266).(U) Genes that confer resistance to Phytophthora Root Rot, such as theRps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7 and other Rps genes.See, for example, Shoemaker, et al., Phytophthora Root Rot ResistanceGene Mapping in Soybean, Plant Genome IV Conference, San Diego, Calif.(1995).(V) Genes that confer resistance to Brown Stem Rot, such as described inU.S. Pat. No. 5,689,035 and incorporated by reference for this purpose.(W) Genes that confer resistance to Colletotrichum, such as described inUS Patent Application Publication US 2009/0035765 and incorporated byreference for this purpose. This includes the Rcg locus that may beutilized as a single locus conversion.2. Transgenes that Confer Resistance to a Herbicide, for Example:(A) A polynucleotide encoding resistance to a herbicide that inhibitsthe growing point or meristem, such as an imidazolinone or asulfonylurea. Exemplary genes in this category code for mutant ALS andAHAS enzyme as described, for example, by Lee, et al., (1988) EMBO J.7:1241 and Miki, et al., (1990) Theor. Appl. Genet. 80:449,respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870;5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937 and5,378,824; U.S. patent application Ser. No. 11/683,737 and InternationalPublication WO 1996/33270.(B) A polynucleotide encoding a protein for resistance to Glyphosate(resistance imparted by mutant 5-enolpyruvl-3-phosphikimate synthase(EPSP) and aroA genes, respectively) and other phosphono compounds suchas glufosinate (phosphinothricin acetyl transferase (PAT) andStreptomyces hygroscopicus phosphinothricin acetyl transferase (bar)genes), and pyridinoxy or phenoxy proprionic acids and cyclohexones(ACCase inhibitor-encoding genes). See, for example, U.S. Pat. No.4,940,835 to Shah, et al., which discloses the nucleotide sequence of aform of EPSPS which can confer glyphosate resistance. U.S. Pat. No.5,627,061 to Barry, et al., also describes genes encoding EPSPS enzymes.See also, U.S. Pat. Nos. 6,566,587; 6,338,961; 6,248,876 B1; 6,040,497;5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642;5,094,945, 4,940,835; 5,866,775; 6,225,114 B1; 6,130,366; 5,310,667;4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E and5,491,288 and International Publications EP 1173580; WO 2001/66704; EP1173581 and EP 1173582, which are incorporated herein by reference forthis purpose. Glyphosate resistance is also imparted to plants thatexpress a gene encoding a glyphosate oxidoreductase enzyme as describedmore fully in U.S. Pat. Nos. 5,776,760 and 5,463,175, which areincorporated herein by reference for this purpose. In additionglyphosate resistance can be imparted to plants by the over expressionof genes encoding glyphosate N-acetyltransferase. See, for example, U.S.Pat. Nos. 7,462,481; 7,405,074 and US Patent Application PublicationNumber US 2008/0234130. A DNA molecule encoding a mutant aroA gene canbe obtained under ATCC® Accession Number 39256, and the nucleotidesequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061 toComai. EP Application Number 0 333 033 to Kumada, et al., and U.S. Pat.No. 4,975,374 to Goodman, et al., disclose nucleotide sequences ofglutamine synthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyltransferase gene is provided in EP ApplicationNumbers 0 242 246 and 0 242 236 to Leemans, et al.; De Greef, et al.,(1989) Bio/Technology 7:61, describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. See also, U.S. Pat. Nos. 5,969,213; 5,489,520;5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024;6,177,616 B1 and 5,879,903, which are incorporated herein by referencefor this purpose. Exemplary genes conferring resistance to phenoxyproprionic acids and cyclohexones, such as sethoxydim and haloxyfop, arethe Acc1-S1, Acc1-S2 and Acc1-S3 genes described by Marshall, et al.,(1992) Theor. Appl. Genet. 83:435.(C) A polynucleotide encoding a protein for resistance to herbicide thatinhibits photosynthesis, such as a triazine (psbA and gs+genes) and abenzonitrile (nitrilase gene). Przibilla, et al., (1991) Plant Cell3:169, describe the transformation of Chlamydomonas with plasmidsencoding mutant psbA genes. Nucleotide sequences for nitrilase genes aredisclosed in U.S. Pat. No. 4,810,648 to Stalker and DNA moleculescontaining these genes are available under ATCC® Accession Numbers53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes, et al., (1992) Biochem.J. 285:173.(D) A polynucleotide encoding a protein for resistance to Acetohydroxyacid synthase, which has been found to make plants that express thisenzyme resistant to multiple types of herbicides, has been introducedinto a variety of plants (see, e.g., Hattori, et al., (1995) Mol GenGenet. 246:419). Other genes that confer resistance to herbicidesinclude: a gene encoding a chimeric protein of rat cytochrome P4507A1and yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., (1994)Plant Physiol 106:17), genes for glutathione reductase and superoxidedismutase (Aono, et al., (1995) Plant Cell Physiol 36:1687) and genesfor various phosphotransferases (Datta, et al., (1992) Plant Mol Biol20:619).(E) A polynucleotide encoding resistance to a herbicide targetingProtoporphyrinogen oxidase (protox) which is necessary for theproduction of chlorophyll. The protox enzyme serves as the target for avariety of herbicidal compounds. These herbicides also inhibit growth ofall the different species of plants present, causing their totaldestruction. The development of plants containing altered protoxactivity which are resistant to these herbicides are described in U.S.Pat. Nos. 6,288,306 B1; 6,282,837 B1 and 5,767,373 and InternationalPublication WO 2001/12825.(F) The aad-1 gene (originally from Sphingobium herbicidovorans) encodesthe aryloxyalkanoate dioxygenase (AAD-1) protein. The trait conferstolerance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate(commonly referred to as “fop” herbicides such as quizalofop)herbicides. The aad-1 gene, itself, for herbicide tolerance in plantswas first disclosed in WO 2005/107437 (see also, US 2009/0093366). Theaad-12 gene, derived from Delftia acidovorans, which encodes thearyloxyalkanoate dioxygenase (AAD-12) protein that confers tolerance to2,4-dichlorophenoxyacetic acid and pyridyloxyacetate herbicides bydeactivating several herbicides with an aryloxyalkanoate moiety,including phenoxy auxin (e.g., 2,4-D, MCPA), as well as pyridyloxyauxins (e.g., fluroxypyr, triclopyr).(G) A polynucleotide encoding a herbicide resistant dicambamonooxygenase disclosed in US Patent Application Publication2003/0135879 for imparting dicamba tolerance;(H) A polynucleotide molecule encoding bromoxynil nitrilase (Bxn)disclosed in U.S. Pat. No. 4,810,648 for imparting bromoxynil tolerance;(I) A polynucleotide molecule encoding phytoene (crtl) described inMisawa, et al., (1993) Plant J. 4:833-840 and in Misawa, et al., (1994)Plant J. 6:481-489 for norflurazon tolerance.3. Transgenes that Confer or Contribute to an Altered GrainCharacteristicSuch as:(A) Altered fatty acids, for example, by(1) Down-regulation of stearoyl-ACP to increase stearic acid content ofthe plant. See, Knultzon, et al., (1992) Proc. Natl. Acad. Sci. USA89:2624 and WO 1999/64579 (Genes to Alter Lipid Profiles in Corn).(2) Elevating oleic acid via FAD-2 gene modification and/or decreasinglinolenic acid via FAD-3 gene modification (see, U.S. Pat. Nos.6,063,947; 6,323,392; 6,372,965 and WO 1993/11245).(3) Altering conjugated linolenic or linoleic acid content, such as inWO 2001/12800.(4) Altering LEC1, AGP, Dek1, Superal1, mi1 ps, various Ipa genes suchas Ipa1, Ipa3, hpt or hggt. For example, see, WO 2002/42424, WO1998/22604, WO 2003/011015, WO 2002/057439, WO 2003/011015, U.S. Pat.Nos. 6,423,886, 6,197,561, 6,825,397 and US Patent ApplicationPublication Numbers US 2003/0079247, US 2003/0204870 and Rivera-Madrid,et al., (1995) Proc. Natl. Acad. Sci. 92:5620-5624.(5) Genes encoding delta-8 desaturase for making long-chainpolyunsaturated fatty acids (U.S. Pat. Nos. 8,058,571 and 8,338,152),delta-9 desaturase for lowering saturated fats (U.S. Pat. No.8,063,269), Primula Δ6-desaturase for improving omega-3 fatty acidprofiles.(6) Isolated nucleic acids and proteins associated with lipid and sugarmetabolism regulation, in particular, lipid metabolism protein (LMP)used in methods of producing transgenic plants and modulating levels ofseed storage compounds including lipids, fatty acids, starches or seedstorage proteins and use in methods of modulating the seed size, seednumber, seed weights, root length and leaf size of plants (EP 2404499).(7) Altering expression of a High-Level Expression of Sugar-Inducible 2(HSI2) protein in the plant to increase or decrease expression of HSI2in the plant. Increasing expression of HSI2 increases oil content whiledecreasing expression of HSI2 decreases abscisic acid sensitivity and/orincreases drought resistance (US Patent Application Publication Number2012/0066794).(8) Expression of cytochrome b5 (Cb5) alone or with FAD2 to modulate oilcontent in plant seed, particularly to increase the levels of omega-3fatty acids and improve the ratio of omega-6 to omega-3 fatty acids (USPatent Application Publication Number 2011/0191904).(9) Nucleic acid molecules encoding wrinkled1-like polypeptides formodulating sugar metabolism (U.S. Pat. No. 8,217,223).(B) Altered phosphorus content, for example, by the(1) Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant. Forexample, see, Van Hartingsveldt, et al., (1993) Gene 127:87, for adisclosure of the nucleotide sequence of an Aspergillus niger phytasegene.(2) Modulating a gene that reduces phytate content. In maize, this, forexample, could be accomplished, by cloning and then re-introducing DNAassociated with one or more of the alleles, such as the LPA alleles,identified in maize mutants characterized by low levels of phytic acid,such as in WO 2005/113778 and/or by altering inositol kinase activity asin WO 2002/059324, US Patent Application Publication Number2003/0009011, WO 2003/027243, US Patent Application Publication Number2003/0079247, WO 1999/05298, U.S. Pat. Nos. 6,197,561, 6,291,224,6,391,348, WO 2002/059324, US Patent Application Publication Number2003/0079247, WO 1998/45448, WO 1999/55882, WO 2001/04147.(C) Altered carbohydrates affected, for example, by altering a gene foran enzyme that affects the branching pattern of starch or, a genealtering thioredoxin such as NTR and/or TRX (see, U.S. Pat. No.6,531,648. which is incorporated by reference for this purpose) and/or agamma zein knock out or mutant such as cs27 or TUSC27 or en27 (see, U.S.Pat. No. 6,858,778 and US Patent Application Publication Number2005/0160488, US Patent Application Publication Number 2005/0204418,which are incorporated by reference for this purpose). See, Shiroza, etal., (1988) J. Bacteriol. 170:810 (nucleotide sequence of Streptococcusmutant fructosyltransferase gene), Steinmetz, et al., (1985) Mol. Gen.Genet. 200:220 (nucleotide sequence of Bacillus subtilis levansucrasegene), Pen, et al., (1992) Bio/Technology 10:292 (production oftransgenic plants that express Bacillus licheniformis alpha-amylase),Elliot, et al., (1993) Plant Molec. Biol. 21:515 (nucleotide sequencesof tomato invertase genes), Segaard, et al., (1993) J. Biol. Chem.268:22480 (site-directed mutagenesis of barley alpha-amylase gene) andFisher, et al., (1993) Plant Physiol. 102:1045 (maize endosperm starchbranching enzyme II), WO 1999/10498 (improved digestibility and/orstarch extraction through modification of UDP-D-xylose 4-epimerase,Fragile 1 and 2, Ref1, HCHL, C4H), U.S. Pat. No. 6,232,529 (method ofproducing high oil seed by modification of starch levels (AGP)). Thefatty acid modification genes mentioned herein may also be used toaffect starch content and/or composition through the interrelationshipof the starch and oil pathways.(D) Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. For example, see, U.S. Pat. No. 6,787,683,US Patent Application Publication Number 2004/0034886 and WO 2000/68393involving the manipulation of antioxidant levels and WO 2003/082899through alteration of a homogentisate geranyl geranyl transferase(hggt).(E) Altered essential seed amino acids. For example, see, U.S. Pat. No.6,127,600 (method of increasing accumulation of essential amino acids inseeds), U.S. Pat. No. 6,080,913 (binary methods of increasingaccumulation of essential amino acids in seeds), U.S. Pat. No. 5,990,389(high lysine), WO 1999/40209 (alteration of amino acid compositions inseeds), WO 1999/29882 (methods for altering amino acid content ofproteins), U.S. Pat. No. 5,850,016 (alteration of amino acidcompositions in seeds), WO 1998/20133 (proteins with enhanced levels ofessential amino acids), U.S. Pat. No. 5,885,802 (high methionine), U.S.Pat. No. 5,885,801 (high threonine), U.S. Pat. No. 6,664,445 (plantamino acid biosynthetic enzymes), U.S. Pat. No. 6,459,019 (increasedlysine and threonine), U.S. Pat. No. 6,441,274 (plant tryptophansynthase beta subunit), U.S. Pat. No. 6,346,403 (methionine metabolicenzymes), U.S. Pat. No. 5,939,599 (high sulfur), U.S. Pat. No. 5,912,414(increased methionine), WO 1998/56935 (plant amino acid biosyntheticenzymes), WO 1998/45458 (engineered seed protein having higherpercentage of essential amino acids), WO 1998/42831 (increased lysine),U.S. Pat. No. 5,633,436 (increasing sulfur amino acid content), U.S.Pat. No. 5,559,223 (synthetic storage proteins with defined structurecontaining programmable levels of essential amino acids for improvementof the nutritional value of plants), WO 1996/01905 (increasedthreonine), WO 1995/15392 (increased lysine), US Patent ApplicationPublication Number 2003/0163838, US Patent Application PublicationNumber 2003/0150014, US Patent Application Publication Number2004/0068767, U.S. Pat. No. 6,803,498, WO 2001/79516.4. Genes that Control Male-Sterility:

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar, et al., and chromosomal translocationsas described by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen, et al., U.S. Pat. No. 5,432,068,describe a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

(A) Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN—Ac-PPT (WO 2001/29237).

(B) Introduction of various stamen-specific promoters (WO 1992/13956, WO1992/13957).

(C) Introduction of the barnase and the barstar gene (Paul, et al.,(1992) Plant Mol. Biol. 19:611-622).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. Nos. 5,859,341; 6,297,426; 5,478,369;5,824,524; 5,850,014 and 6,265,640, all of which are hereby incorporatedby reference.

5. Genes that Create a Site for Site Specific DNA Integration.

This includes the introduction of FRT sites that may be used in theFLP/FRT system and/or Lox sites that may be used in the Cre/Loxp system.For example, see, Lyznik, et al., (2003) Plant Cell Rep 21:925-932 andWO 1999/25821, which are hereby incorporated by reference. Other systemsthat may be used include the Gln recombinase of phage Mu (Maeser, etal., (1991) Vicki Chandler, The Maize Handbook ch. 118 (Springer-Verlag1994), the Pin recombinase of E. coli (Enomoto, et al., 1983) and theR/RS system of the pSRi plasmid (Araki, et al., 1992).6. Genes that Affect Abiotic Stress ResistanceIncluding but not limited to flowering, ear and seed development,enhancement of nitrogen utilization efficiency, altered nitrogenresponsiveness, drought resistance or tolerance, cold resistance ortolerance and salt resistance or tolerance and increased yield understress.(A) For example, see: WO 2000/73475 where water use efficiency isaltered through alteration of malate; U.S. Pat. Nos. 5,892,009,5,965,705, 5,929,305, 5,891,859, 6,417,428, 6,664,446, 6,706,866,6,717,034, 6,801,104, WO 2000/060089, WO 2001/026459, WO 2001/035725, WO2001/034726, WO 2001/035727, WO 2001/036444, WO 2001/036597, WO2001/036598, WO 2002/015675, WO 2002/017430, WO 2002/077185, WO2002/079403, WO 2003/013227, WO 2003/013228, WO 2003/014327, WO2004/031349, WO 2004/076638, WO 199809521.(B) WO 199938977 describing genes, including CBF genes and transcriptionfactors effective in mitigating the negative effects of freezing, highsalinity and drought on plants, as well as conferring other positiveeffects on plant phenotype.(C) US Patent Application Publication Number 2004/0148654 and WO2001/36596 where abscisic acid is altered in plants resulting inimproved plant phenotype such as increased yield and/or increasedtolerance to abiotic stress.(D) WO 2000/006341, WO 2004/090143, U.S. Pat. Nos. 7,531,723 and6,992,237 where cytokinin expression is modified resulting in plantswith increased stress tolerance, such as drought tolerance, and/orincreased yield. Also see, WO 2002/02776, WO 2003/052063, JP2002/281975, U.S. Pat. No. 6,084,153, WO 2001/64898, U.S. Pat. Nos.6,177,275 and 6,107,547 (enhancement of nitrogen utilization and alterednitrogen responsiveness).(E) For ethylene alteration, see, US Patent Application PublicationNumber 2004/0128719, US Patent Application Publication Number2003/0166197 and WO 2000/32761.(F) For plant transcription factors or transcriptional regulators ofabiotic stress, see, e.g., US Patent Application Publication Number2004/0098764 or US Patent Application Publication Number 2004/0078852.(G) Genes that increase expression of vacuolar pyrophosphatase such asAVP1 (U.S. Pat. No. 8,058,515) for increased yield; nucleic acidencoding a HSFA4 or a HSFA5 (Heat Shock Factor of the class A4 or A5)polypeptides, an oligopeptide transporter protein (OPT4-like)polypeptide; a plastochron2-like (PLA2-like) polypeptide or a Wuschelrelated homeobox 1-like (WOX1-like) polypeptide (U. Patent ApplicationPublication Number US 2011/0283420).(H) Down regulation of polynucleotides encoding poly (ADP-ribose)polymerase (PARP) proteins to modulate programmed cell death (U.S. Pat.No. 8,058,510) for increased vigor.(I) Polynucleotide encoding DTP21 polypeptides for conferring droughtresistance (US Patent Application Publication Number US 2011/0277181).(J) Nucleotide sequences encoding ACC Synthase 3 (ACS3) proteins formodulating development, modulating response to stress, and modulatingstress tolerance (US Patent Application Publication Number US2010/0287669).(K) Polynucleotides that encode proteins that confer a drought tolerancephenotype (DTP) for conferring drought resistance (WO 2012/058528).(L) Tocopherol cyclase (TC) genes for conferring drought and salttolerance (US Patent Application Publication Number 2012/0272352).(M) CAAX amino terminal family proteins for stress tolerance (U.S. Pat.No. 8,338,661).(N) Mutations in the SAL1 encoding gene have increased stress tolerance,including increased drought resistant (US Patent Application PublicationNumber 2010/0257633).(O) Expression of a nucleic acid sequence encoding a polypeptideselected from the group consisting of: GRF polypeptide, RAA1-likepolypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptideincreasing yield-related traits (US Patent Application PublicationNumber 2011/0061133).(P) Modulating expression in a plant of a nucleic acid encoding a ClassIII Trehalose Phosphate Phosphatase (TPP) polypeptide for enhancingyield-related traits in plants, particularly increasing seed yield (USPatent Application Publication Number 2010/0024067).Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, can be introduced or introgressed into plants, see e.g., WO1997/49811 (LHY), WO 1998/56918 (ESD4), WO 1997/10339 and U.S. Pat. No.6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO 1996/14414 (CON), WO1996/38560, WO 2001/21822 (VRN1), WO 2000/44918 (VRN2), WO 1999/49064(GI), WO 2000/46358 (FR1), WO 1997/29123, U.S. Pat. Nos. 6,794,560,6,307,126 (GAI), WO 1999/09174 (D8 and Rht) and WO 2004/076638 and WO2004/031349 (transcription factors).7. Genes that Confer Increased Yield(A) A transgenic crop plant transformed by a1-AminoCyclopropane-1-Carboxylate Deaminase-like Polypeptide (ACCDP)coding nucleic acid, wherein expression of the nucleic acid sequence inthe crop plant results in the plant's increased root growth, and/orincreased yield, and/or increased tolerance to environmental stress ascompared to a wild type variety of the plant (U.S. Pat. No. 8,097,769).(B) Over-expression of maize zinc finger protein gene (Zm-ZFP1) using aseed preferred promoter has been shown to enhance plant growth, increasekernel number and total kernel weight per plant (US Patent ApplicationPublication Number 2012/0079623).(C) Constitutive over-expression of maize lateral organ boundaries (LOB)domain protein (Zm-LOBDP1) has been shown to increase kernel number andtotal kernel weight per plant (US Patent Application Publication Number2012/0079622).(D) Enhancing yield-related traits in plants by modulating expression ina plant of a nucleic acid encoding a VIM1 (Variant in Methylation1)-like polypeptide or a VTC2-like (GDP-L-galactose phosphorylase)polypeptide or a DUF1685 polypeptide or an ARF6-like (Auxin ResponsiveFactor) polypeptide (WO 2012/038893).(E) Modulating expression in a plant of a nucleic acid encoding aSte20-like polypeptide or a homologue thereof gives plants havingincreased yield relative to control plants (EP 2431472).(F) Genes encoding nucleoside diphosphatase kinase (NDK) polypeptidesand homologs thereof for modifying the plant's root architecture (USPatent Application Publication Number 2009/0064373).8. Genes that Confer Plant Digestibility.(A) Altering the level of xylan present in the cell wall of a plant bymodulating expression of xylan synthase (U.S. Pat. No. 8,173,866).In some embodiment the stacked trait may be a trait or event that hasreceived regulatory approval including but not limited to the events inTable 5A-5F.

TABLE 5A Medicago sativa Alfalfa Event Company Description J101,Monsanto Glyphosate herbicide tolerant alfalfa (lucerne) J163 Companyand produced by inserting a gene encoding the Forage Genetics enzyme5-enolypyruvylshikimate-3-phosphate International synthase (EPSPS) fromthe CP4 strain of Agrobacterium tumefaciens.

TABLE 5B Helianthus annuus Sunflower Event Company Description X81359BASF Inc. Tolerance to imidazolinone herbicides by selection of anaturally occurring mutant.

TABLE 5C Oryza sativa Rice Event Company Description CL121, BASF Inc.Tolerance to the imidazolinone herbicide, CL141, imazethapyr, induced bychemical mutagenesis of CFX51 the acetolactate synthase (ALS) enzymeusing ethyl methanesulfonate (EMS). IMINTA-1, BASF Inc. Tolerance toimidazolinone herbicides induced by IMINTA-4 chemical mutagenesis of theacetolactate synthase (ALS) enzyme using sodium azide. LLRICE06, AventisCropScience Glufosinate ammonium herbicide tolerant rice LLRICE62produced by inserting a modified phosphinothricin acetyltransferase(PAT) encoding gene from the soil bacterium Streptomyces hygroscopicus).LLRICE601 Bayer CropScience Glufosinate ammonium herbicide tolerant rice(Aventis produced by inserting a modified phosphinothricinCropScience(AgrEvo)) acetyltransferase (PAT) encoding gene from the soilbacterium Streptomyces hygroscopicus). PWC16 BASF Inc. Tolerance to theimidazolinone herbicide, imazethapyr, induced by chemical mutagenesis ofthe acetolactate synthase (ALS) enzyme using ethyl methanesulfonate(EMS).

TABLE 5C Triticum aestivum Wheat Event Company Description AP205CL BASFInc. Selection for a mutagenized version of the enzyme acetohydroxyacidsynthase (AHAS), also known as acetolactate synthase (ALS) oracetolactate pyruvate-lyase. AP602CL BASF Inc. Selection for amutagenized version of the enzyme acetohydroxyacid synthase (AHAS), alsoknown as acetolactate synthase (ALS) or acetolactate pyruvate-lyase.BW255-2, BASF Inc. Selection for a mutagenized version of the enzymeBW238-3 acetohydroxyacid synthase (AHAS), also known as acetolactatesynthase (ALS) or acetolactate pyruvate-lyase. BW7 BASF Inc. Toleranceto imidazolinone herbicides induced by chemical mutagenesis of theacetohydroxyacid synthase (AHAS) gene using sodium azide. MON71800Monsanto Company Glyphosate tolerant wheat variety produced by insertinga modified 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encodinggene from the soil bacterium Agrobacterium tumefaciens, strain CP4.SWP965001 Cyanamid Crop Selection for a mutagenized version of theenzyme Protection acetohydroxyacid synthase (AHAS), also known asacetolactate synthase (ALS) or acetolactate pyruvate-lyase. Teal 11ABASF Inc. Selection for a mutagenized version of the enzymeacetohydroxyacid synthase (AHAS), also known as acetolactate synthase(ALS) or acetolactate pyruvate-lyase.

TABLE 5E Glycine max L. Soybean Event Company Description A2704-12,Bayer CropScience Glufosinate ammonium herbicide tolerant soybeanA2704-21, (Aventis CropScience produced by inserting a modifiedphosphinothricin A5547-35 (AgrEvo)) acetyltransferase (PAT) encodinggene from the soil bacterium Streptomyces viridochromogenes. A5547-127Bayer CropScience Glufosinate ammonium herbicide tolerant soybean(Aventis CropScience produced by inserting a modified phosphinothricin(AgrEvo)) acetyltransferase (PAT) encoding gene from the soil bacteriumStreptomyces viridochromogenes. BPS-CV127-9 BASF Inc. The introducedcsr1-2 gene from Arabidopsis thaliana encodes an acetohydroxyacidsynthase protein that confers tolerance to imidazolinone herbicides dueto a point mutation that results in a single amino acid substitution inwhich the serine residue at position 653 is replaced by asparagine(S653N). DP-305423 Pioneer Hi-Bred High oleic acid soybean produced byinserting International Inc. additional copies of a portion of theomega-6 desaturase encoding gene, gm-fad2-1 resulting in silencing ofthe endogenous omega-6 desaturase gene (FAD2-1). DP356043 PioneerHi-Bred Soybean event with two herbicide tolerance International Inc.genes: glyphosate N-acetlytransferase, which detoxifies glyphosate, anda modified acetolactate synthase (ALS) gene which is tolerant to ALS-inhibiting herbicides. G94-1, G94-19, DuPont Canada High oleic acidsoybean produced by inserting a G168 Agricultural Products second copyof the fatty acid desaturase (GmFad2-1) encoding gene from soybean,which resulted in “silencing” of the endogenous host gene. GTS 40-3-2Monsanto Company Glyphosate tolerant soybean variety produced byinserting a modified 5-enolpyruvylshikimate-3- phosphate synthase(EPSPS) encoding gene from the soil bacterium Agrobacterium tumefaciens.GU262 Bayer CropScience Glufosinate ammonium herbicide tolerant soybean(Aventis produced by inserting a modified phosphinothricinCropScience(AgrEvo)) acetyltransferase (PAT) encoding gene from the soilbacterium Streptomyces viridochromogenes. MON87701 Monsanto CompanyResistance to Lepidopteran pests of soybean including velvetbeancaterpillar (Anticarsia gemmatalis) and soybean looper (Pseudoplusiaincludens). MON87701 × Monsanto Company Glyphosate herbicide tolerancethrough MON89788 expression of the EPSPS encoding gene from A.tumefaciens strain CP4, and resistance to Lepidopteran pests of soybeanincluding velvetbean caterpillar (Anticarsia gemmatalis) and soybeanlooper (Pseudoplusia includens) via expression of the Cry1Ac encodinggene from B. thuringiensis. MON89788 Monsanto CompanyGlyphosate-tolerant soybean produced by inserting a modified5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encoding aroA(epsps) gene from Agrobacterium tumefaciens CP4. OT96-15 Agriculture &Agri-Food Low linolenic acid soybean produced through Canada traditionalcross-breeding to incorporate the novel trait from a naturally occurringfan1 gene mutant that was selected for low linolenic acid. W62, W98Bayer CropScience Glufosinate ammonium herbicide tolerant soybean(Aventis produced by inserting a modified phosphinothricinCropScience(AgrEvo)) acetyltransferase (PAT) encoding gene from the soilbacterium Streptomyces hygroscopicus.

TABLE 5F Zea mays L. Maize Event Company Description 176 Syngenta Seeds,Inc. Insect-resistant maize produced by inserting the Cry1Ab gene fromBacillus thuringiensis subsp. kurstaki. The genetic modification affordsresistance to attack by the European corn borer (ECB). 3751IR PioneerHi-Bred Selection of somaclonal variants by culture of InternationalInc. embryos on imidazolinone containing media. 676, 678, 680 PioneerHi-Bred Male-sterile and glufosinate ammonium herbicide InternationalInc. tolerant maize produced by inserting genes encoding DNA adeninemethylase and phosphinothricin acetyltransferase (PAT) from Escherichiacoli and Streptomyces viridochromogenes, respectively. B16 (DLL25)Dekalb Genetics Glufosinate ammonium herbicide tolerant maizeCorporation produced by inserting the gene encoding phosphinothricinacetyltransferase (PAT) from Streptomyces hygroscopicus. BT11 (X4334CBR,Syngenta Seeds, Inc. Insect-resistant and herbicide tolerant maizeX4734CBR) produced by inserting the Cry1Ab gene from Bacillusthuringiensis subsp. kurstaki, and the phosphinothricinN-acetyltransferase (PAT) encoding gene from S. viridochromogenes. BT11× GA21 Syngenta Seeds, Inc. Stacked insect resistant and herbicidetolerant maize produced by conventional cross breeding of parental linesBT11 (OECD unique identifier: SYN- BTO11-1) and GA21 (OECD uniqueidentifier: MON-OOO21-9). BT11 × MIR162 × Syngenta Seeds, Inc.Resistance to Coleopteran pests, particularly corn MIR604 × GA21rootworm pests (Diabrotica spp.) and several Lepidopteran pests of corn,including European corn borer (ECB, Ostrinia nubilalis), corn earworm(CEW, Helicoverpa zea), fall army worm (FAW, Spodoptera frugiperda), andblack cutworm (BCW, Agrotis ipsilon); tolerance to glyphosate andglufosinate-ammonium containing herbicides. BT11 × MIR162 SyngentaSeeds, Inc. Stacked insect resistant and herbicide tolerant maizeproduced by conventional cross breeding of parental lines BT11 (OECDunique identifier: SYN- BTO11-1) and MIR162 (OECD unique identifier:SYN-IR162-4). Resistance to the European Corn Borer and tolerance to theherbicide glufosinate ammonium (Liberty) is derived from BT11, whichcontains the Cry1Ab gene from Bacillus thuringiensis subsp. kurstaki,and the phosphinothricin N-acetyltransferase (PAT) encoding gene from S.viridochromogenes. Resistance to other Lepidopteran pests, including H.zea, S. frugiperda, A. ipsilon, and S. albicosta, is derived fromMIR162, which contains the vip3Aa gene from Bacillus thuringiensisstrain AB88. BT11 × MIR162 × Syngenta Seeds, Inc. Bacillus thuringiensisCry1Ab delta-endotoxin MIR604 protein and the genetic material necessaryfor its production (via elements of vector pZO1502) in Event Bt11 corn(OECD Unique Identifier: SYN- BTO11-1) x Bacillus thuringiensis Vip3Aa20insecticidal protein and the genetic material necessary for itsproduction (via elements of vector pNOV1300) in Event MIR162 maize (OECDUnique Identifier: SYN-IR162-4) x modified Cry3A protein and the geneticmaterial necessary for its production (via elements of vector pZM26) inEvent MIR604 corn (OECD Unique Identifier: SYN-IR6O4-5). CBH-351 AventisCropScience Insect-resistant and glufosinate ammonium herbicide tolerantmaize developed by inserting genes encoding Cry9C protein from Bacillusthuringiensis subsp tolworthi and phosphinothricin acetyltransferase(PAT) from Streptomyces hygroscopicus. DAS-06275-8 DOW AgroSciences LLCLepidopteran insect resistant and glufosinate ammoniumherbicide-tolerant maize variety produced by inserting the Cry1F genefrom Bacillus thuringiensis var aizawai and the phosphinothricinacetyltransferase (PAT) from Streptomyces hygroscopicus. BT11 × MIR604Syngenta Seeds, Inc. Stacked insect resistant and herbicide tolerantmaize produced by conventional cross breeding of parental lines BT11(OECD unique identifier: SYN- BTO11-1) and MIR604 (OECD uniqueidentifier: SYN-IR6O5-5). Resistance to the European Corn Borer andtolerance to the herbicide glufosinate ammonium (Liberty) is derivedfrom BT11, which contains the Cry1Ab gene from Bacillus thuringiensissubsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT)encoding gene from S. viridochromogenes. Corn rootworm-resistance isderived from MIR604 which contains the mCry3A gene from Bacillusthuringiensis. BT11 × MIR604 × Syngenta Seeds, Inc. Stacked insectresistant and herbicide tolerant GA21 maize produced by conventionalcross breeding of parental lines BT11 (OECD unique identifier: SYN-BTO11-1), MIR604 (OECD unique identifier: SYN- IR6O5-5) and GA21 (OECDunique identifier: MON-OOO21-9). Resistance to the European Corn Borerand tolerance to the herbicide glufosinate ammonium (Liberty) is derivedfrom BT11, which contains the Cry1Ab gene from Bacillus thuringiensissubsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT)encoding gene from S. viridochromogenes. Corn rootworm-resistance isderived from MIR604 which contains the mCry3A gene from Bacillusthuringiensis. Tolerance to glyphosate herbicide is derived from GA21which contains a a modified EPSPS gene from maize. DAS-59122-7 DOWAgroSciences LLC Corn rootworm-resistant maize produced by and PioneerHi-Bred inserting the Cry34Ab1 and Cry35Ab1 genes from InternationalInc. Bacillus thuringiensis strain PS149B1. The PAT encoding gene fromStreptomyces viridochromogenes was introduced as a selectable marker.DAS-59122-7 × DOW AgroSciences LLC Stacked insect resistant andherbicide tolerant TC1507× NK603 and Pioneer Hi-Bred maize produced byconventional cross breeding of International Inc. parental linesDAS-59122-7 (OECD unique identifier: DAS-59122-7) and TC1507 (OECDunique identifier: DAS-O15O7-1) with NK603 (OECD unique identifier:MON-OO6O3-6). Corn rootworm-resistance is derived from DAS-59122-7 whichcontains the Cry34Ab1 and Cry35Ab1 genes from Bacillus thuringiensisstrain PS149B1. Lepidopteran resistance and tolerance to glufosinateammonium herbicide is derived from TC1507. Tolerance to glyphosateherbicide is derived from NK603. DBT418 Dekalb Genetics Insect-resistantand glufosinate ammonium Corporation herbicide tolerant maize developedby inserting genes encoding Cry1AC protein from Bacillus thuringiensissubsp kurstaki and phosphinothricin acetyltransferase (PAT) fromStreptomyces hygroscopicus MIR604 × GA21 Syngenta Seeds, Inc. Stackedinsect resistant and herbicide tolerant maize produced by conventionalcross breeding of parental lines MIR604 (OECD unique identifier:SYN-IR6O5-5) and GA21 (OECD unique identifier: MON-OOO21-9). Cornrootworm-resistance is derived from MIR604 which contains the mCry3Agene from Bacillus thuringiensis. Tolerance to glyphosate herbicide isderived from GA21. MON80100 Monsanto Company Insect-resistant maizeproduced by inserting the Cry1Ab gene from Bacillus thuringiensis subsp.kurstaki. The genetic modification affords resistance to attack by theEuropean corn borer (ECB). MON802 Monsanto Company Insect-resistant andglyphosate herbicide tolerant maize produced by inserting the genesencoding the Cry1Ab protein from Bacillus thuringiensis and the5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from A. tumefaciensstrain CP4. MON809 Pioneer Hi-Bred Resistance to European corn borer(Ostrinia International Inc. nubilalis) by introduction of a syntheticCry1Ab gene. Glyphosate resistance via introduction of the bacterialversion of a plant enzyme, 5-enolpyruvyl shikimate-3-phosphate synthase(EPSPS). MON810 Monsanto Company Insect-resistant maize produced byinserting a truncated form of the Cry1Ab gene from Bacillusthuringiensis subsp. kurstaki HD-1. The genetic modification affordsresistance to attack by the European corn borer (ECB). MON810 × LY038Monsanto Company Stacked insect resistant and enhanced lysine contentmaize derived from conventional cross- breeding of the parental linesMON810 (OECD identifier: MON-OO810-6) and LY038 (OECD identifier:REN-OOO38-3). MON810 × MON88017 Monsanto Company Stacked insectresistant and glyphosate tolerant maize derived from conventionalcross-breeding of the parental lines MON810 (OECD identifier:MON-OO81O-6) and MON88017 (OECD identifier: MON-88O17-3). European cornborer (ECB) resistance is derived from a truncated form of the Cry1Abgene from Bacillus thuringiensis subsp. kurstaki HD-1 present in MON810.Corn rootworm resistance is derived from the Cry3Bb1 gene from Bacillusthuringiensis subspecies kumamotoensis strain EG4691 present inMON88017. Glyphosate tolerance is derived from a5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene fromAgrobacterium tumefaciens strain CP4 present in MON88017. MON832Monsanto Company Introduction, by particle bombardment, of glyphosateoxidase (GOX) and a modified 5- enolpyruvyl shikimate-3-phosphatesynthase (EPSPS), an enzyme involved in the shikimate biochemicalpathway for the production of the aromatic amino acids. MON863 MonsantoCompany Corn rootworm resistant maize produced by inserting the Cry3Bb1gene from Bacillus thuringiensis subsp. kumamotoensis. MON863 × MON810Monsanto Company Stacked insect resistant corn hybrid derived fromconventional cross-breeding of the parental lines MON863 (OECDidentifier: MON-OO863-5) and MON810 (OECD identifier: MON-OO81O-6)MON863 × MON810 × Monsanto Company Stacked insect resistant andherbicide tolerant NK603 corn hybrid derived from conventional cross-breeding of the stacked hybrid MON-OO863-5 x MON-OO81O-6 and NK603 (OECDidentifier: MON-OO6O3-6). MON863 × NK603 Monsanto Company Stacked insectresistant and herbicide tolerant corn hybrid derived from conventionalcross- breeding of the parental lines MON863 (OECD identifier:MON-OO863-5) and NK603 (OECD identifier: MON-OO6O3-6). MON87460 MonsantoCompany MON 87460 was developed to provide reduced yield loss underwater-limited conditions compared to conventional maize. Efficacy in MON87460 is derived by expression of the inserted Bacillus subtilis coldshock protein B (CspB). MON88017 Monsanto Company Cornrootworm-resistant maize produced by inserting the Cry3Bb1 gene fromBacillus thuringiensis subspecies kumamotoensis strain EG4691.Glyphosate tolerance derived by inserting a5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene fromAgrobacterium tumefaciens strain CP4. MON89034 Monsanto Company Maizeevent expressing two different insecticidal proteins from Bacillusthuringiensis providing resistance to number of Lepidopteran pests.MON89034 × Monsanto Company Stacked insect resistant and glyphosatetolerant MON88017 maize derived from conventional cross-breeding of theparental lines MON89034 (OECD identifier: MON-89O34-3) and MON88017(OECD identifier: MON-88O17-3). Resistance to Lepidopteran insects isderived from two Cry genes present in MON89043. Corn rootworm resistanceis derived from a single Cry genes and glyphosate tolerance is derivedfrom the 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS) encodinggene from Agrobacterium tumefaciens present in MON88017. MON89034 ×NK603 Monsanto Company Stacked insect resistant and herbicide tolerantmaize produced by conventional cross breeding of parental lines MON89034(OECD identifier: MON- 89O34-3) with NK603 (OECD unique identifier:MON-OO6O3-6). Resistance to Lepidopteran insects is derived from two Crygenes present in MON89043. Tolerance to glyphosate herbicide is derivedfrom NK603. NK603 × Monsanto Company Stacked insect resistant andherbicide tolerant corn MON810 hybrid derived from conventionalcross-breeding of the parental lines NK603 (OECD identifier:MON-OO6O3-6) and MON810 (OECD identifier: MON-OO81O-6). MON89034 ×Monsanto Company and Stacked insect resistant and herbicide tolerantmaize TC1507 × Mycogen Seeds c/o Dow produced by conventional crossbreeding of parental MON88017 × AgroSciences LLC lines: MON89034,TC1507, MON88017, and DAS- DAS-59122-7 59122. Resistance to theabove-ground and below- ground insect pests and tolerance to glyphosateand glufosinate-ammonium containing herbicides. MS3 Bayer CropScienceMale sterility caused by expression of the barnase (Aventis ribonucleasegene from Bacillus amyloliquefaciens; PPT CropScience(AgrEvo))resistance was via PPT-acetyltransferase (PAT). MS6 Bayer CropScienceMale sterility caused by expression of the barnase (Aventis ribonucleasegene from Bacillus amyloliquefaciens; PPT CropScience(AgrEvo))resistance was via PPT-acetyltransferase (PAT). NK603 Monsanto CompanyIntroduction, by particle bombardment, of a modified 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme involved in theshikimate biochemical pathway for the production of the aromatic aminoacids. NK603 × T25 Monsanto Company Stacked glufosinate ammonium andglyphosate herbicide tolerant maize hybrid derived from conventionalcross-breeding of the parental lines NK603 (OECD identifier:MON-OO6O3-6) and T25 (OECD identifier: ACS-ZM003-2). T25 × MON810 BayerCropScience Stacked insect resistant and herbicide tolerant corn(Aventis hybrid derived from conventional cross-breeding of theCropScience(AgrEvo)) parental lines T25 (OECD identifier: ACS-ZMOO3-2)and MON810 (OECD identifier: MON-OO81O-6). TC1507 Mycogen (c/o DowInsect-resistant and glufosinate ammonium herbicide AgroSciences);Pioneer tolerant maize produced by inserting the Cry1F gene (c/o DuPont)from Bacillus thuringiensis var. aizawai and the phosphinothricinN-acetyltransferase encoding gene from Streptomyces viridochromogenes.TC1507 × DOW AgroSciences LLC Stacked insect resistant and herbicidetolerant corn NK603 hybrid derived from conventional cross-breeding ofthe parental lines 1507 (OECD identifier: DAS-O15O7-1) and NK603 (OECDidentifier: MON-OO6O3-6). TC1507 × DAS- DOW AgroSciences LLC Stackedinsect resistant and herbicide tolerant maize 59122-7 and PioneerHi-Bred produced by conventional cross breeding of parentalInternational Inc. lines TC1507 (OECD unique identifier: DAS-O15O7-1)with DAS-59122-7 (OECD unique identifier: DAS-59122- 7). Resistance toLepidopteran insects is derived from TC1507 due the presence of theCry1F gene from Bacillus thuringiensis var. aizawai. Corn rootworm-resistance is derived from DAS-59122-7 which contains the Cry34Ab1 andCry35Ab1 genes from Bacillus thuringiensis strain PS149B1. Tolerance toglufosinate ammonium herbicide is derived from TC1507 from thephosphinothricin N-acetyltransferase encoding gene from Streptomycesviridochromogenes.Other events with regulatory approval are well known to one skilled inthe art and can be found at the Center for Environmental Risk Assessment(cera-gmc.org/?action=gm_crop_database, which can be accessed using thewww prefix) and at the International Service for the Acquisition ofAgri-Biotech Applications (isaaa.org/gmapprovaldatabase/default.asp,which can be accessed using the www prefix).Gene SilencingIn some embodiments the stacked trait may be in the form of silencing ofone or more polynucleotides of interest resulting in suppression of oneor more target pest polypeptides. In some embodiments the silencing isachieved through the use of a suppression DNA construct. In someembodiments one or more polynucleotide encoding the polypeptides of thePtIP-83 polypeptide or fragments or variants thereof may be stacked withone or more polynucleotides encoding one or more polypeptides havinginsecticidal activity or agronomic traits as set forth supra andoptionally may further include one or more polynucleotides providing forgene silencing of one or more target polynucleotides as discussed infra.“Suppression DNA construct” is a recombinant DNA construct which whentransformed or stably integrated into the genome of the plant, resultsin “silencing” of a target gene in the plant. The target gene may beendogenous or transgenic to the plant. “Silencing,” as used herein withrespect to the target gene, refers generally to the suppression oflevels of mRNA or protein/enzyme expressed by the target gene, and/orthe level of the enzyme activity or protein functionality. The term“suppression” includes lower, reduce, decline, decrease, inhibit,eliminate and prevent. “Silencing” or “gene silencing” does not specifymechanism and is inclusive, and not limited to, anti-sense,cosuppression, viral-suppression, hairpin suppression, stem-loopsuppression, RNAi-based approaches and small RNA-based approaches.

A suppression DNA construct may comprise a region derived from a targetgene of interest and may comprise all or part of the nucleic acidsequence of the sense strand (or antisense strand) of the target gene ofinterest. Depending upon the approach to be utilized, the region may be100% identical or less than 100% identical (e.g., at least 50% or anyinteger between 51% and 100% identical) to all or part of the sensestrand (or antisense strand) of the gene of interest.

Suppression DNA constructs are well-known in the art, are readilyconstructed once the target gene of interest is selected, and include,without limitation, cosuppression constructs, antisense constructs,viral-suppression constructs, hairpin suppression constructs, stem-loopsuppression constructs, double-stranded RNA-producing constructs, andmore generally, RNAi (RNA interference) constructs and small RNAconstructs such as siRNA (short interfering RNA) constructs and miRNA(microRNA) constructs.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.

“Antisense RNA” refers to an RNA transcript that is complementary to allor part of a target primary transcript or mRNA and that blocks theexpression of a target isolated nucleic acid fragment (U.S. Pat. No.5,107,065). The complementarity of an antisense RNA may be with any partof the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, introns or the coding sequence.

“Cosuppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of the target protein. “Sense” RNArefers to RNA transcript that includes the mRNA and can be translatedinto protein within a cell or in vitro. Cosuppression constructs inplants have been previously designed by focusing on overexpression of anucleic acid sequence having homology to a native mRNA, in the senseorientation, which results in the reduction of all RNA having homologyto the overexpressed sequence (see, Vaucheret, et al., (1998) Plant J.16:651-659 and Gura, (2000) Nature 404:804-808).

Another variation describes the use of plant viral sequences to directthe suppression of proximal mRNA encoding sequences (PCT Publication WO1998/36083).

Recent work has described the use of “hairpin” structures thatincorporate all or part, of an mRNA encoding sequence in a complementaryorientation that results in a potential “stem-loop” structure for theexpressed RNA (PCT Publication WO 1999/53050). In this case the stem isformed by polynucleotides corresponding to the gene of interest insertedin either sense or anti-sense orientation with respect to the promoterand the loop is formed by some polynucleotides of the gene of interest,which do not have a complement in the construct. This increases thefrequency of cosuppression or silencing in the recovered transgenicplants. For review of hairpin suppression, see, Wesley, et al., (2003)Methods in Molecular Biology, Plant Functional Genomics: Methods andProtocols 236:273-286.

A construct where the stem is formed by at least 30 nucleotides from agene to be suppressed and the loop is formed by a random nucleotidesequence has also effectively been used for suppression (PCT PublicationWO 1999/61632).

The use of poly-T and poly-A sequences to generate the stem in thestem-loop structure has also been described (PCT Publication WO2002/00894).

Yet another variation includes using synthetic repeats to promoteformation of a stem in the stem-loop structure. Transgenic organismsprepared with such recombinant DNA fragments have been shown to havereduced levels of the protein encoded by the nucleotide fragment formingthe loop as described in PCT Publication WO 2002/00904.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire, et al., (1998) Nature 391:806). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing (PTGS) or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire, et al., (1999) TrendsGenet. 15:358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single-stranded RNA of viral genomicRNA. The presence of dsRNA in cells triggers the RNAi response through amechanism that has yet to be fully characterized.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein, et al., (2001) Nature 409:363).Short interfering RNAs derived from dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes (Elbashir, et al., (2001) Genes Dev. 15:188). Dicer has alsobeen implicated in the excision of 21- and 22-nucleotide small temporalRNAs (stRNAs) from precursor RNA of conserved structure that areimplicated in translational control (Hutvagner, et al., (2001) Science293:834). The RNAi response also features an endonuclease complex,commonly referred to as an RNA-induced silencing complex (RISC), whichmediates cleavage of single-stranded RNA having sequence complementarityto the antisense strand of the siRNA duplex. Cleavage of the target RNAtakes place in the middle of the region complementary to the antisensestrand of the siRNA duplex (Elbashir, et al., (2001) Genes Dev. 15:188).In addition, RNA interference can also involve small RNA (e.g., miRNA)mediated gene silencing, presumably through cellular mechanisms thatregulate chromatin structure and thereby prevent transcription of targetgene sequences (see, e.g., Allshire, (2002) Science 297:1818-1819;Volpe, et al., (2002) Science 297:1833-1837; Jenuwein, (2002) Science297:2215-2218 and Hall, et al., (2002) Science 297:2232-2237). As such,miRNA molecules of the disclosure can be used to mediate gene silencingvia interaction with RNA transcripts or alternately by interaction withparticular gene sequences, wherein such interaction results in genesilencing either at the transcriptional or post-transcriptional level.

Methods and compositions are further provided which allow for anincrease in RNAi produced from the silencing element. In suchembodiments, the methods and compositions employ a first polynucleotidecomprising a silencing element for a target pest sequence operablylinked to a promoter active in the plant cell; and, a secondpolynucleotide comprising a suppressor enhancer element comprising thetarget pest sequence or an active variant or fragment thereof operablylinked to a promoter active in the plant cell. The combined expressionof the silencing element with suppressor enhancer element leads to anincreased amplification of the inhibitory RNA produced from thesilencing element over that achievable with only the expression of thesilencing element alone. In addition to the increased amplification ofthe specific RNAi species itself, the methods and compositions furtherallow for the production of a diverse population of RNAi species thatcan enhance the effectiveness of disrupting target gene expression. Assuch, when the suppressor enhancer element is expressed in a plant cellin combination with the silencing element, the methods and compositioncan allow for the systemic production of RNAi throughout the plant; theproduction of greater amounts of RNAi than would be observed with justthe silencing element construct alone; and, the improved loading of RNAiinto the phloem of the plant, thus providing better control of phloemfeeding insects by an RNAi approach. Thus, the various methods andcompositions provide improved methods for the delivery of inhibitory RNAto the target organism. See, for example, US Patent ApplicationPublication 2009/0188008.

As used herein, a “suppressor enhancer element” comprises apolynucleotide comprising the target sequence to be suppressed or anactive fragment or variant thereof. It is recognize that the suppressorenhancer element need not be identical to the target sequence, butrather, the suppressor enhancer element can comprise a variant of thetarget sequence, so long as the suppressor enhancer element hassufficient sequence identity to the target sequence to allow for anincreased level of the RNAi produced by the silencing element over thatachievable with only the expression of the silencing element. Similarly,the suppressor enhancer element can comprise a fragment of the targetsequence, wherein the fragment is of sufficient length to allow for anincreased level of the RNAi produced by the silencing element over thatachievable with only the expression of the silencing element.

It is recognized that multiple suppressor enhancer elements from thesame target sequence or from different target sequences or fromdifferent regions of the same target sequence can be employed. Forexample, the suppressor enhancer elements employed can comprisefragments of the target sequence derived from different region of thetarget sequence (i.e., from the 3′UTR, coding sequence, intron, and/or5′UTR). Further, the suppressor enhancer element can be contained in anexpression cassette, as described elsewhere herein, and in specificembodiments, the suppressor enhancer element is on the same or on adifferent DNA vector or construct as the silencing element. Thesuppressor enhancer element can be operably linked to a promoter asdisclosed herein. It is recognized that the suppressor enhancer elementcan be expressed constitutively or alternatively, it may be produced ina stage-specific manner employing the various inducible ortissue-preferred or developmentally regulated promoters that arediscussed elsewhere herein.

In specific embodiments, employing both a silencing element and thesuppressor enhancer element the systemic production of RNAi occursthroughout the entire plant. In further embodiments, the plant or plantparts of the disclosure have an improved loading of RNAi into the phloemof the plant than would be observed with the expression of the silencingelement construct alone and, thus provide better control of phloemfeeding insects by an RNAi approach. In specific embodiments, theplants, plant parts and plant cells of the disclosure can further becharacterized as allowing for the production of a diversity of RNAispecies that can enhance the effectiveness of disrupting target geneexpression.

In specific embodiments, the combined expression of the silencingelement and the suppressor enhancer element increases the concentrationof the inhibitory RNA in the plant cell, plant, plant part, plant tissueor phloem over the level that is achieved when the silencing element isexpressed alone.

As used herein, an “increased level of inhibitory RNA” comprises anystatistically significant increase in the level of RNAi produced in aplant having the combined expression when compared to an appropriatecontrol plant. For example, an increase in the level of RNAi in theplant, plant part or the plant cell can comprise at least about a 1%,about a 1%-5%, about a 5%-10%, about a 10%-20%, about a 20%-30%, about a30%-40%, about a 40%-50%, about a 50%-60%, about 60-70%, about 70%-80%,about a 80%-90%, about a 90%-100% or greater increase in the level ofRNAi in the plant, plant part, plant cell or phloem when compared to anappropriate control. In other embodiments, the increase in the level ofRNAi in the plant, plant part, plant cell or phloem can comprise atleast about a 1 fold, about a 1 fold-5 fold, about a 5 fold-10 fold,about a 10 fold-20 fold, about a 20 fold-30 fold, about a 30 fold-40fold, about a 40 fold-50 fold, about a 50 fold-60 fold, about 60 fold-70fold, about 70 fold-80 fold, about a 80 fold-90 fold, about a 90fold-100 fold or greater increase in the level of RNAi in the plant,plant part, plant cell or phloem when compared to an appropriatecontrol. Examples of combined expression of the silencing element withsuppressor enhancer element for the control of Stinkbugs and Lygus canbe found in US Patent Application Publication 2011/0301223 and US PatentApplication Publication 2009/0192117.

Some embodiments relate to down-regulation of expression of target genesin insect pest species by interfering ribonucleic acid (RNA) molecules.PCT Publication WO 2007/074405 describes methods of inhibitingexpression of target genes in invertebrate pests including Coloradopotato beetle. PCT Publication WO 2005/110068 describes methods ofinhibiting expression of target genes in invertebrate pests including inparticular Western corn rootworm as a means to control insectinfestation. Furthermore, PCT Publication WO 2009/091864 describescompositions and methods for the suppression of target genes from insectpest species including pests from the Lygus genus. Nucleic acidmolecules including RNAi for targeting the vacuolar ATPase H subunit,useful for controlling a coleopteran pest population and infestation asdescribed in US Patent Application Publication 2012/0198586. PCTPublication WO 2012/055982 describes ribonucleic acid (RNA or doublestranded RNA) that inhibits or down regulates the expression of a targetgene that encodes: an insect ribosomal protein such as the ribosomalprotein L19, the ribosomal protein L40 or the ribosomal protein S27A; aninsect proteasome subunit such as the Rpn6 protein, the Pros 25, theRpn2 protein, the proteasome beta 1 subunit protein or the Pros beta 2protein; an insect β-coatomer of the COPI vesicle, the γ-coatomer of theCOPI vesicle, the β′-coatomer protein or the ζ-coatomer of the COPIvesicle; an insect Tetraspanine 2 A protein which is a putativetransmembrane domain protein; an insect protein belonging to the actinfamily such as Actin 5C; an insect ubiquitin-5E protein; an insect Sec23protein which is a GTPase activator involved in intracellular proteintransport; an insect crinkled protein which is an unconventional myosinwhich is involved in motor activity; an insect crooked neck proteinwhich is involved in the regulation of nuclear alternative mRNAsplicing; an insect vacuolar H+-ATPase G-subunit protein and an insectTbp-1 such as Tat-binding protein. US Patent Application Publications2012/029750, US 20120297501, and 2012/0322660 describe interferingribonucleic acids (RNA or double stranded RNA) that functions uponuptake by an insect pest species to down-regulate expression of a targetgene in said insect pest, wherein the RNA comprises at least onesilencing element wherein the silencing element is a region ofdouble-stranded RNA comprising annealed complementary strands, onestrand of which comprises or consists of a sequence of nucleotides whichis at least partially complementary to a target nucleotide sequencewithin the target gene. US Patent Application Publication 2012/0164205describe potential targets for interfering double stranded ribonucleicacids for inhibiting invertebrate pests including: a Chd3 HomologousSequence, a Beta-Tubulin Homologous Sequence, a 40 kDa V-ATPaseHomologous Sequence, a EF1α Homologous Sequence, a 26S ProteosomeSubunit p28 Homologous Sequence, a Juvenile Hormone Epoxide HydrolaseHomologous Sequence, a Swelling Dependent Chloride Channel ProteinHomologous Sequence, a Glucose-6-Phosphate 1-Dehydrogenase ProteinHomologous Sequence, an Act42A Protein Homologous Sequence, aADP-Ribosylation Factor 1 Homologous Sequence, a Transcription FactorIIB Protein Homologous Sequence, a Chitinase Homologous Sequences, aUbiquitin Conjugating Enzyme Homologous Sequence, aGlyceraldehyde-3-Phosphate Dehydrogenase Homologous Sequence, anUbiquitin B Homologous Sequence, a Juvenile Hormone Esterase Homolog,and an Alpha Tubuliln Homologous Sequence.

Use in Pesticidal Control

General methods for employing strains comprising a nucleic acid sequenceof the embodiments or a variant thereof, in pesticide control or inengineering other organisms as pesticidal agents are known in the art.See, for example U.S. Pat. No. 5,039,523 and EP 0480762A2.

Microorganism hosts that are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one ormore crops of interest may be selected. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment with the wild-type microorganisms, provide for stablemaintenance and expression of the gene expressing the PtIP-83polypeptide and desirably provide for improved protection of thepesticide from environmental degradation and inactivation.

Such microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms such as bacteria, e.g., Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus,Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungi,particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interestare such phytosphere bacterial species as Pseudomonas syringae,Pseudomonas fluorescens, Pseudomonas chlororaphis, Serratia marcescens,Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides,Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus,Clavibacter xyli and Azotobacter vinelandii and phytosphere yeastspecies such as Rhodotorula rubra, R. glutinis, R. marina, R.aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomycesroseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans.Of particular interest are the pigmented microorganisms. Host organismsof particular interest include yeast, such as Rhodotorula spp.,Aureobasidium spp., Saccharomyces spp. (such as S. cerevisiae),Sporobolomyces spp., phylloplane organisms such as Pseudomonas spp.(such as P. aeruginosa, P. fluorescens, P. chlororaphis), Erwinia spp.,and Flavobacterium spp., and other such organisms, includingAgrobacterium tumefaciens, E. coli, Bacillus subtilis, Bacillus cereusand the like.

Genes encoding the PtIP-83 polypeptide of the embodiments can beintroduced into microorganisms that multiply on plants (epiphytes) todeliver PtIP-83 polypeptide to potential target pests. Epiphytes, forexample, can be gram-positive or gram-negative bacteria.

Root-colonizing bacteria, for example, can be isolated from the plant ofinterest by methods known in the art. Specifically, a Bacillus cereusstrain that colonizes roots can be isolated from roots of a plant (see,for example, Handelsman et al. (1991) Appl. Environ. Microbiol.56:713-718). Genes encoding the PtIP-83 polypeptide of the embodimentscan be introduced into a root-colonizing Bacillus cereus by standardmethods known in the art.

Genes encoding PtIP-83 polypeptides can be introduced, for example, intothe root-colonizing Bacillus by means of electro transformation.Specifically, genes encoding the PtIP-83 polypeptides can be cloned intoa shuttle vector, for example, pHT3101 (Lerecius, et al., (1989) FEMSMicrobiol. Letts. 60:211-218. The shuttle vector pHT3101 containing thecoding sequence for the particular PtIP-83 polypeptide gene can, forexample, be transformed into the root-colonizing Bacillus by means ofelectroporation (Lerecius, et al., (1989) FEMS Microbiol. Letts.60:211-218).

Expression systems can be designed so that PtIP-83 polypeptides aresecreted outside the cytoplasm of gram-negative bacteria, such as E.coli, for example. Advantages of having a PtIP-83 polypeptide secretedare: (1) avoidance of potential cytotoxic effects of the PtIP-83polypeptide expressed; and (2) improvement in the efficiency ofpurification of the PtIP-83 polypeptide, including, but not limited to,increased efficiency in the recovery and purification of the protein pervolume cell broth and decreased time and/or costs of recovery andpurification per unit protein.

PtIP-83 polypeptides can be made to be secreted in E. coli, for example,by fusing an appropriate E. coli signal peptide to the amino-terminalend of the PtIP-83 polypeptide. Signal peptides recognized by E. colican be found in proteins already known to be secreted in E. coli, forexample the OmpA protein (Ghrayeb, et al., (1984) EMBO J, 3:2437-2442).OmpA is a major protein of the E. coli outer membrane, and thus itssignal peptide is thought to be efficient in the translocation process.Also, the OmpA signal peptide does not need to be modified beforeprocessing as may be the case for other signal peptides, for examplelipoprotein signal peptide (Duffaud, et al., (1987) Meth. Enzymol.153:492).

PtIP-83 polypeptides of the embodiments can be fermented in a bacterialhost and the resulting bacteria processed and used as a microbial sprayin the same manner that Bt strains have been used as insecticidalsprays. In the case of a PtIP-83 polypeptide(s) that is secreted fromBacillus, the secretion signal is removed or mutated using proceduresknown in the art. Such mutations and/or deletions prevent secretion ofthe PtIP-83 polypeptide(s) into the growth medium during thefermentation process. The PtIP-83 polypeptide is retained within thecell, and the cells are then processed to yield the encapsulated PtIP-83polypeptide. Any suitable microorganism can be used for this purpose.Pseudomonas has been used to express Bt toxins as encapsulated proteinsand the resulting cells processed and sprayed as an insecticide(Gaertner, et al., (1993), in: Advanced Engineered Pesticides, ed. Kim).

Alternatively, the PtIP-83 polypeptide is produced by introducing aheterologous gene into a cellular host. Expression of the heterologousgene results, directly or indirectly, in the intracellular productionand maintenance of the pesticide. These cells are then treated underconditions that prolong the activity of the toxin produced in the cellwhen the cell is applied to the environment of target pest(s). Theresulting product retains the toxicity of the toxin. These naturallyencapsulated PtIP-83 polypeptide may then be formulated in accordancewith conventional techniques for application to the environment hostinga target pest, e.g., soil, water, and foliage of plants. See, forexample EPA 0192319, and the references cited therein.

Pesticidal Compositions

In some embodiments the active ingredients can be applied in the form ofcompositions and can be applied to the crop area or plant to be treated,simultaneously or in succession, with other compounds. These compoundscan be fertilizers, weed killers, Cryoprotectants, surfactants,detergents, pesticidal soaps, dormant oils, polymers, and/ortime-release or biodegradable carrier formulations that permit long-termdosing of a target area following a single application of theformulation. They can also be selective herbicides, chemicalinsecticides, virucides, microbicides, amoebicides, pesticides,fungicides, bacteriocides, nematocides, molluscicides or mixtures ofseveral of these preparations, if desired, together with furtheragriculturally acceptable carriers, surfactants or application-promotingadjuvants customarily employed in the art of formulation. Suitablecarriers and adjuvants can be solid or liquid and correspond to thesubstances ordinarily employed in formulation technology, e.g. naturalor regenerated mineral substances, solvents, dispersants, wettingagents, tackifiers, binders or fertilizers. Likewise the formulationsmay be prepared into edible “baits” or fashioned into pest “traps” topermit feeding or ingestion by a target pest of the pesticidalformulation.

Methods of applying an active ingredient or an agrochemical compositionthat contains at least one of the PtIP-83 polypeptide produced by thebacterial strains include leaf application, seed coating and soilapplication. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding pest.

The composition may be formulated as a powder, dust, pellet, granule,spray, emulsion, colloid, solution or such like, and may be prepared bysuch conventional means as desiccation, lyophilization, homogenation,extraction, filtration, centrifugation, sedimentation or concentrationof a culture of cells comprising the polypeptide. In all suchcompositions that contain at least one such pesticidal polypeptide, thepolypeptide may be present in a concentration of from about 1% to about99% by weight.

Lepidopteran, Dipteran, Heteropteran, nematode, Hemiptera or Coleopteranpests may be killed or reduced in numbers in a given area by the methodsof the disclosure or may be prophylactically applied to an environmentalarea to prevent infestation by a susceptible pest. Preferably the pestingests or is contacted with, a pesticidally-effective amount of thepolypeptide. “Pesticidally-effective amount” as used herein refers to anamount of the pesticide that is able to bring about death to at leastone pest or to noticeably reduce pest growth, feeding or normalphysiological development. This amount will vary depending on suchfactors as, for example, the specific target pests to be controlled, thespecific environment, location, plant, crop or agricultural site to betreated, the environmental conditions and the method, rate,concentration, stability, and quantity of application of thepesticidally-effective polypeptide composition. The formulations mayalso vary with respect to climatic conditions, environmentalconsiderations, and/or frequency of application and/or severity of pestinfestation.

The pesticide compositions described may be made by formulating eitherthe bacterial cell, Crystal and/or spore suspension or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, desiccated or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial or a suspension in oil (vegetable or mineral) or water oroil/water emulsions or as a wettable powder or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,inert components, dispersants, surfactants, tackifiers, binders, etc.that are ordinarily used in pesticide formulation technology; these arewell known to those skilled in pesticide formulation. The formulationsmay be mixed with one or more solid or liquid adjuvants and prepared byvarious means, e.g., by homogeneously mixing, blending and/or grindingthe pesticidal composition with suitable adjuvants using conventionalformulation techniques. Suitable formulations and application methodsare described in U.S. Pat. No. 6,468,523, herein incorporated byreference. The plants can also be treated with one or more chemicalcompositions, including one or more herbicide, insecticides orfungicides. Exemplary chemical compositions include: Fruits/VegetablesHerbicides: Atrazine, Bromacil, Diuron, Glyphosate, Linuron, Metribuzin,Simazine, Trifluralin, Fluazifop, Glufosinate, Halo sulfuron Gowan,Paraquat, Propyzamide, Sethoxydim, Butafenacil, Halosulfuron,Indaziflam; Fruits/Vegetables Insecticides: Aldicarb, Bacillusthuriengiensis, Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin,Deltamethrin, Diazinon, Malathion, Abamectin,Cyfluthrin/beta-cyfluthrin, Esfenvalerate, Lambda-cyhalothrin,Acequinocyl, Bifenazate, Methoxyfenozide, Novaluron, Chromafenozide,Thiacloprid, Dinotefuran, FluaCrypyrim, Tolfenpyrad, Clothianidin,Spirodiclofen, Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr,Cyazypyr, Spinoteram, Triflumuron, Spirotetramat, Imidacloprid,Flubendiamide, Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumetofen,Cyanopyrafen, Imidacloprid, Clothianidin, Thiamethoxam, Spinotoram,Thiodicarb, Flonicamid, Methiocarb, Emamectin-benzoate, Indoxacarb,Forthiazate, Fenamiphos, Cadusaphos, Pyriproxifen, Fenbutatin-oxid,Hexthiazox, Methomyl,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on;Fruits/Vegetables Fungicides: Carbendazim, Chlorothalonil, EBDCs,Sulphur, Thiophanate-methyl, Azoxystrobin, Cymoxanil, Fluazinam,Fosetyl, Iprodione, Kresoxim-methyl, Metalaxyl/mefenoxam,Trifloxystrobin, Ethaboxam, Iprovalicarb, Trifloxystrobin, Fenhexamid,Oxpoconazole fumarate, Cyazofamid, Fenamidone, Zoxamide, Picoxystrobin,Pyraclostrobin, Cyflufenamid, Boscalid; Cereals Herbicides: Isoproturon,Bromoxynil, loxynil, Phenoxies, Chlorsulfuron, Clodinafop, Diclofop,Diflufenican, Fenoxaprop, Florasulam, Fluoroxypyr, Metsulfuron,Triasulfuron, Flucarbazone, lodosulfuron, Propoxycarbazone, Picolinafen,Mesosulfuron, Beflubutamid, Pinoxaden, Amidosulfuron, ThifensulfuronMethyl, Tribenuron, Flupyrsulfuron, Sulfosulfuron, Pyrasulfotole,Pyroxsulam, Flufenacet, Tralkoxydim, Pyroxasulfon; Cereals Fungicides:Carbendazim, Chlorothalonil, Azoxystrobin, Cyproconazole, Cyprodinil,Fenpropimorph, Epoxiconazole, Kresoxim-methyl, Quinoxyfen, Tebuconazole,Trifloxystrobin, Simeconazole, Picoxystrobin, Pyraclostrobin,Dimoxystrobin, Prothioconazole, Fluoxastrobin; Cereals Insecticides:Dimethoate, Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin,β-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos, Metamidophos,Oxidemethon-methyl, Pirimicarb, Methiocarb; Maize Herbicides: Atrazine,Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S-)Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor,Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione,Foramsulfuron, Topramezone, Tembotrione, Saflufenacil, Thiencarbazone,Flufenacet, Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyrifos,Bifenthrin, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin,Terbufos, Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide,Triflumuron, Rynaxypyr, Deltamethrin, Thiodicarb, β-Cyfluthrin,Cypermethrin, Bifenthrin, Lufenuron, Triflumoron, Tefluthrin,Tebupirimphos, Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid,Dinetofuran, Avermectin, Methiocarb, Spirodiclofen, Spirotetramat; MaizeFungicides: Fenitropan, Thiram, Prothioconazole, Tebuconazole,Trifloxystrobin; Rice Herbicides: Butachlor, Propanil, Azimsulfuron,Bensulfuron, Cyhalofop, Daimuron, Fentrazamide, Imazosulfuron,Mefenacet, Oxaziclomefone, Pyrazosulfuron, Pyributicarb, Quinclorac,Thiobencarb, Indanofan, Flufenacet, Fentrazamide, Halosulfuron,Oxaziclomefone, Benzobicyclon, Pyriftalid, Penoxsulam, Bispyribac,Oxadiargyl, Ethoxysulfuron, Pretilachlor, Mesotrione, Tefuryltrione,Oxadiazone, Fenoxaprop, Pyrimisulfan; Rice Insecticides: Diazinon,Fenitrothion, Fenobucarb, Monocrotophos, Benfuracarb, Buprofezin,Dinotefuran, Fipronil, Imidacloprid, Isoprocarb, Thiacloprid,Chromafenozide, Thiacloprid, Dinotefuran, Clothianidin, Ethiprole,Flubendiamide, Rynaxypyr, Deltamethrin, Acetamiprid, Thiamethoxam,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Cypermethrin,Chlorpyriphos, Cartap, Methamidophos, Etofenprox, Triazophos,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Carbofuran, Benfuracarb; Rice Fungicides: Thiophanate-methyl,Azoxystrobin, Carpropamid, Edifenphos, Ferimzone, Iprobenfos,Isoprothiolane, Pencycuron, Probenazole, Pyroquilon, Tricyclazole,Trifloxystrobin, Diclocymet, Fenoxanil, Simeconazole, Tiadinil; CottonHerbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn,Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate,Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron,Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; CottonInsecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin,Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid,Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin,Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl,Flonicamid, Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin,Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran,Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen,Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton Fungicides:Etridiazole, Metalaxyl, Quintozene; Soybean Herbicides: Alachlor,Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl,Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin,Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim,Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl,Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole,Deltamethrin, β-Cyfluthrin, gamma and lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb,beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Cyproconazole,Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole,Trifloxystrobin, Prothioconazole, Tetraconazole; Sugarbeet Herbicides:Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate,Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim,Triflusulfuron, Tepraloxydim, Quizalofop; Sugarbeet Insecticides:Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid,Dinetofuran, Deltamethrin, β-Cyfluthrin, gamma/lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran; CanolaHerbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate,Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop,Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin, Carbendazim,Fludioxonil, Iprodione, Prochloraz, Vinclozolin; Canola Insecticides:Carbofuran organophosphates, Pyrethroids, Thiacloprid, Deltamethrin,Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran,β-Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole,Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on.

In some embodiments the herbicide is Atrazine, Bromacil, Diuron,Chlorsulfuron, Metsulfuron, Thifensulfuron Methyl, Tribenuron,Acetochlor, Dicamba, Isoxaflutole, Nicosulfuron, Rimsulfuron,Pyrithiobac-sodium, Flumioxazin, Chlorimuron-Ethyl, Metribuzin,Quizalofop, S-metolachlor, Hexazinne or combinations thereof.

In some embodiments the insecticide is Esfenvalerate,Chlorantraniliprole, Methomyl, Indoxacarb, Oxamyl or combinationsthereof.

Pesticidal and Insecticidal Activity

“Pest” includes but is not limited to, insects, fungi, bacteria,nematodes, mites, ticks and the like. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyLepidoptera and Coleoptera.

Those skilled in the art will recognize that not all compounds areequally effective against all pests. Compounds of the embodimentsdisplay activity against insect pests, which may include economicallyimportant agronomic, forest, greenhouse, nursery ornamentals, food andfiber, public and animal health, domestic and commercial structure,household and stored product pests.

Larvae of the order Lepidoptera include, but are not limited to,armyworms, cutworms, loopers and heliothines in the family NoctuidaeSpodoptera frugiperda JE Smith (fall armyworm); S. exigua Hübner (beetarmyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar);Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus(cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogoniaMorrison (western cutworm); A. subterranea Fabricius (granulatecutworm); Alabama argillacea Hübner (cotton leaf worm); Trichoplusia niHübner (cabbage looper); Pseudoplusia includens Walker (soybean looper);Anticarsia gemmatalis Hübner (velvetbean caterpillar); Hypena scabraFabricius (green cloverworm); Heliothis virescens Fabricius (tobaccobudworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindaraBarnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris(darksided cutworm); Earias insulana Boisduval (spiny bollworm); E.vittella Fabricius (spotted bollworm); Helicoverpa armigera Hübner(American bollworm); H. zea Boddie (corn earworm or cotton bollworm);Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialisGrote (citrus cutworm); borers, casebearers, webworms, coneworms, andskeletonizers from the family Pyralidae Ostrinia nubilalis Hübner(European corn borer); Amyelois transitella Walker (naval orangeworm);Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautellaWalker (almond moth); Chilo suppressalis Walker (rice stem borer); C.partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth);Crambus caliginosellus Clemens (corn root webworm); C. teterrellusZincken (bluegrass webworm); Cnaphalocrocis medinalis Guenée (rice leafroller); Desmia funeralis Hübner (grape leaffolder); Diaphania hyalinataLinnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraeagrandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius(surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestiaelutella Hübner (tobacco (cacao) moth); Galleria mellonella Linnaeus(greater wax moth); Herpetogramma licarsisalis Walker (sod webworm);Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellusZeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser waxmoth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalisWalker (tea tree web moth); Maruca testulalis Geyer (bean pod borer);Plodia interpunctella Hübner (Indian meal moth); Scirpophaga incertulasWalker (yellow stem borer); Udea rubigalis Guenée (celery leaftier); andleafrollers, budworms, seed worms and fruit worms in the familyTortricidae Acleris gloverana Walsingham (Western blackheaded budworm);A. variana Fernald (Eastern blackheaded budworm); Archips argyrospilaWalker (fruit tree leaf roller); A. rosana Linnaeus (European leafroller); and other Archips species, Adoxophyes orana Fischer vonRösslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham(banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C.pomonella Linnaeus (coding moth); Platynota flavedana Clemens(variegated leafroller); P. stultana Walsingham (omnivorous leafroller);Lobesia botrana Denis & Schiffermüller (European grape vine moth);Spilonota ocellana Denis & Schiffermller (eyespotted bud moth); Endopizaviteana Clemens (grape berry moth); Eupoecilia ambiguella Hübner (vinemoth); Bonagota salubricola Meyrick (Brazilian apple leafroller);Grapholita molesta Busck (oriental fruit moth); Suleima helianthanaRiley (sunflower bud moth); Argyrotaenia spp.; Choristoneura spp.

Selected other agronomic pests in the order Lepidoptera include, but arenot limited to, Alsophila pometaria Harris (fall cankerworm); Anarsialineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith(orange striped oakworm); Antheraea pernyi Guérin-Méneville (Chinese OakTussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiellaBusck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfacaterpillar); Datana integerrima Grote & Robinson (walnut caterpillar);Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomossubsignaria Hübner (elm spanworm); Erannis tiliaria Harris (lindenlooper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisinaamericana Guérin-Méneville (grapeleaf skeletonizer); Hemileuca oliviaeCockrell (range caterpillar); Hyphantria cunea Drury (fall webworm);Keiferia lycopersicella Walsingham (tomato pinworm); Lambdinafiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellarialugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus(satin moth); Lymantria dispar Linnaeus (gypsy moth); Manducaquinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M.sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumataLinnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm);Papilio cresphontes Cramer (giant swallowtail orange dog); Phryganidiacalifornica Packard (California oakworm); Phyllocnistis citrellaStainton (citrus leafminer); Phyllonorycter blancardella Fabricius(spotted tentiform leafminer); Pieris brassicae Linnaeus (large whitebutterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus(green veined white butterfly); Platyptilia carduidactyla Riley(artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth);Pectinophora gossypiella Saunders (pink bollworm); Pontia protodiceBoisduval and Leconte (Southern cabbageworm); Sabulodes aegrotata Guenée(omnivorous looper); Schizura concinna J. E. Smith (red humpedcaterpillar); Sitotroga cerealella Olivier (Angoumois grain moth);Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar);Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick(tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothissubflexa Guenée; Malacosoma spp. and Orgyia spp.

Of interest are larvae and adults of the order Coleoptera includingweevils from the families Anthribidae, Bruchidae and Curculionidae(including, but not limited to: Anthonomus grandis Boheman (bollweevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil);Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (riceweevil); Hypera punctata Fabricius (clover leaf weevil);Cylindrocopturus adspersus LeConte (sunflower stem weevil); Smicronyxfulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (graysunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug));flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetlesand leafminers in the family Chrysomelidae (including, but not limitedto: Leptinotarsa decemlineata Say (Colorado potato beetle); Diabroticavirgifera virgifera LeConte (western corn rootworm); D. barberi Smithand Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber(southern corn rootworm); Chaetocnema pulicaria Melsheimer (corn fleabeetle); Phyllotreta cruciferae Goeze (Crucifer flea beetle);Phyllotreta striolata (stripped flea beetle); Colaspis brunnea Fabricius(grape colaspis); Oulema melanopus Linnaeus (cereal leaf beetle);Zygogramma exclamationis Fabricius (sunflower beetle)); beetles from thefamily Coccinellidae (including, but not limited to: Epilachnavarivestis Mulsant (Mexican bean beetle)); chafers and other beetlesfrom the family Scarabaeidae (including, but not limited to: Popilliajaponica Newman (Japanese beetle); Cyclocephala borealis Arrow (northernmasked chafer, white grub); C. immaculata Olivier (southern maskedchafer, white grub); Rhizotrogus majalis Razoumowsky (European chafer);Phyllophaga crinita Burmeister (white grub); Ligyrus gibbosus De Geer(carrot beetle)); carpet beetles from the family Dermestidae; wirewormsfrom the family Elateridae, Eleodes spp., Melanotus spp.; Conoderusspp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; barkbeetles from the family Scolytidae and beetles from the familyTenebrionidae.

Adults and immatures of the order Diptera are of interest, includingleafminers Agromyza parvicornis Loew (corn blotch leafminer); midges(including, but not limited to: Contarinia sorghicola Coquillett(sorghum midge); Mayetiola destructor Say (Hessian fly); Sitodiplosismosellana Géhin (wheat midge); Neolasioptera murtfeldtiana Felt,(sunflower seed midge)); fruit flies (Tephritidae), Oscinella fritLinnaeus (fruit flies); maggots (including, but not limited to: Deliaplatura Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulb fly)and other Delia spp., Meromyza americana Fitch (wheat stem maggot);Musca domestica Linnaeus (house flies); Fannia canicularis Linnaeus, F.femoralis Stein (lesser house flies); Stomoxys calcitrans Linnaeus(stable flies)); face flies, horn flies, blow flies, Chrysomya spp.;Phormia spp. and other muscoid fly pests, horse flies Tabanus spp.; botflies Gastrophilus spp.; Oestrus spp.; cattle grubs Hypoderma spp.; deerflies Chrysops spp.; Melophagus ovinus Linnaeus (keds) and otherBrachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; blackflies Prosimulium spp.; Simulium spp.; biting midges, sand flies,sciarids, and other Nematocera.

Included as insects of interest are adults and nymphs of the ordersHemiptera and Homoptera such as, but not limited to, adelgids from thefamily Adelgidae, plant bugs from the family Miridae, cicadas from thefamily Cicadidae, leafhoppers, Empoasca spp.; from the familyCicadellidae, planthoppers from the families Cixiidae, Flatidae,Fulgoroidea, Issidae and Delphacidae, treehoppers from the familyMembracidae, psyllids from the family Psyllidae, whiteflies from thefamily Aleyrodidae, aphids from the family Aphididae, phylloxera fromthe family Phylloxeridae, mealybugs from the family Pseudococcidae,scales from the families Asterolecanidae, Coccidae, Dactylopiidae,Diaspididae, Eriococcidae Ortheziidae, Phoenicococcidae andMargarodidae, lace bugs from the family Tingidae, stink bugs from thefamily Pentatomidae, cinch bugs, Blissus spp.; and other seed bugs fromthe family Lygaeidae, spittlebugs from the family Cercopidae squash bugsfrom the family Coreidae and red bugs and cotton stainers from thefamily Pyrrhocoridae.

Agronomically important members from the order Homoptera furtherinclude, but are not limited to: Acyrthisiphon pisum Harris (pea aphid);Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black beanaphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicisForbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecolaPatch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid);Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxiaKurdjumov/Mordvilko (Russian wheat aphid); Dysaphis plantagineaPaaserini (rosy apple aphid); Eriosoma lanigerum Hausmann (woolly appleaphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopteruspruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnipaphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphumeuphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach-potatoaphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid);Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch(corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphisgraminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcaneaphid); Sitobion avenae Fabricius (English grain aphid); Therioaphismaculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer deFonscolombe (black citrus aphid) and T. citricida Kirkaldy (brown citrusaphid); Adelges spp. (adelgids); Phylloxera devastatrix Pergande (pecanphylloxera); Bemisia tabaci Gennadius (tobacco whitefly, sweetpotatowhitefly); B. argentifolii Bellows & Perring (silverleaf whitefly);Dialeurodes citri Ashmead (citrus whitefly); Trialeurodes abutiloneus(bandedwinged whitefly) and T. vaporariorum Westwood (greenhousewhitefly); Empoasca fabae Harris (potato leafhopper); Laodelphaxstriatellus Fallen (smaller brown planthopper); Macrolestesquadrilineatus Forbes (aster leafhopper); Nephotettix cinticeps Uhler(green leafhopper); N. nigropictus Stål (rice leafhopper); Nilaparvatalugens Stål (brown planthopper); Peregrinus maidis Ashmead (cornplanthopper); Sogatella furcifera Horvath (white-backed planthopper);Sogatodes orizicola Muir (rice delphacid); Typhlocyba pomaria McAtee(white apple leafhopper); Erythroneoura spp. (grape leafhoppers);Magicicada septendecim Linnaeus (periodical cicada); Icerya purchasiMaskell (cottony cushion scale); Quadraspidiotus perniciosus Comstock(San Jose scale); Planococcus citri Risso (citrus mealybug);Pseudococcus spp. (other mealybug complex); Cacopsylla pyricola Foerster(pear psylla); Trioza diospyri Ashmead (persimmon psylla).

Agronomically important species of interest from the order Hemipterainclude, but are not limited to: Acrosternum hilare Say (green stinkbug); Anasa tristis De Geer (squash bug); Blissus leucopterusleucopterus Say (chinch bug); Corythuca gossypii Fabricius (cotton lacebug); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellusHerrich-Schäffer (cotton stainer); Euschistus servus Say (brown stinkbug); E. variolarius Palisot de Beauvois (one-spotted stink bug);Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say(leaf-footed pine seed bug); Lygus lineolaris Palisot de Beauvois(tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug);L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius(European tarnished plant bug); Lygocoris pabulinus Linnaeus (commongreen capsid); Nezara viridula Linnaeus (southern green stink bug);Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas(large milkweed bug); Pseudatomoscelis seriatus Reuter (cottonfleahopper).

Furthermore, embodiments may be effective against Hemiptera such,Calocoris norvegicus Gmelin (strawberry bug); Orthops campestrisLinnaeus; Plesiocoris rugicollis Fallen (apple capsid); Cyrtopeltismodestus Distant (tomato bug); Cyrtopeltis notatus Distant (suckfly);Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Diaphnocorischlorionis Say (honeylocust plant bug); Labopidicola allii Knight (onionplant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper);Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatusFabricius (four-lined plant bug); Nysius ericae Schilling (false chinchbug); Nysius raphanus Howard (false chinch bug); Nezara viridulaLinnaeus (Southern green stink bug); Eurygaster spp.; Coreidae spp.;Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidae spp.and Cimicidae spp.

Also included are adults and larvae of the order Acari (mites) such asAceria tosichella Keifer (wheat curl mite); Petrobia latens Müller(brown wheat mite); spider mites and red mites in the familyTetranychidae, Panonychus ulmi Koch (European red mite); Tetranychusurticae Koch (two spotted spider mite); (T. mcdanieli McGregor (McDanielmite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestaniUgarov & Nikolski (strawberry spider mite); flat mites in the familyTenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust andbud mites in the family Eriophyidae and other foliar feeding mites andmites important in human and animal health, i.e., dust mites in thefamily Epidermoptidae, follicle mites in the family Demodicidae, grainmites in the family Glycyphagidae, ticks in the order Ixodidae: Ixodesscapularis Say (deer tick); I. holocyclus Neumann (Australian paralysistick); Dermacentor variabilis Say (American dog tick); Amblyommaamericanum Linnaeus (lone star tick) and scab and itch mites in thefamilies Psoroptidae, Pyemotidae and Sarcoptidae.

Insect pests of the order Thysanura are of interest, such as Lepismasaccharina Linnaeus (silverfish); Thermobia domestica Packard(firebrat).

Additional arthropod pests covered include: spiders in the order Araneaesuch as Loxosceles reclusa Gertsch and Mulaik (brown recluse spider) andthe Latrodectus mactans Fabricius (black widow spider) and centipedes inthe order Scutigeromorpha such as Scutigera coleoptrata Linnaeus (housecentipede).

Insect pest of interest include the superfamily of stink bugs and otherrelated insects including but not limited to species belonging to thefamily Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorusguildini, Euschistus servus, Acrosternum hilare, Euschistus heros,Euschistus tristigmus, Acrosternum hilare, Dichelops furcatus, Dichelopsmelacanthus, and Bagrada hilaris (Bagrada Bug)), the family Plataspidae(Megacopta cribraria—Bean plataspid) and the family Cydnidae(Scaptocoris castanea—Root stink bug) and Lepidoptera species includingbut not limited to: diamond-back moth, e.g., Helicoverpa zea Boddie;soybean looper, e.g., Pseudoplusia includens Walker and velvet beancaterpillar e.g., Anticarsia gemmatalis Hübner.

Methods for measuring pesticidal activity are well known in the art.See, for example, Czapla and Lang, (1990) J. Econ. Entomol.83:2480-2485; Andrews, et al., (1988) Biochem. J. 252:199-206; Marrone,et al., (1985) J. of Economic Entomology 78:290-293 and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety. Generally, the protein is mixed and used in feeding assays.See, for example Marrone, et al., (1985) J. of Economic Entomology78:290-293. Such assays can include contacting plants with one or morepests and determining the plant's ability to survive and/or cause thedeath of the pests.

Nematodes include parasitic nematodes such as root-knot, cyst and lesionnematodes, including Heterodera spp., Meloidogyne spp. and Globoderaspp.; particularly members of the cyst nematodes, including, but notlimited to, Heterodera glycines (soybean cyst nematode); Heteroderaschachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode)and Globodera rostochiensis and Globodera pailida (potato cystnematodes). Lesion nematodes include Pratylenchus spp.

Seed Treatment

To protect and to enhance yield production and trait technologies, seedtreatment options can provide additional crop plan flexibility and costeffective control against insects, weeds and diseases. Seed material canbe treated, typically surface treated, with a composition comprisingcombinations of chemical or biological herbicides, herbicide safeners,insecticides, fungicides, germination inhibitors and enhancers,nutrients, plant growth regulators and activators, bactericides,nematocides, avicides and/or molluscicides. These compounds aretypically formulated together with further carriers, surfactants orapplication-promoting adjuvants customarily employed in the art offormulation. The coatings may be applied by impregnating propagationmaterial with a liquid formulation or by coating with a combined wet ordry formulation. Examples of the various types of compounds that may beused as seed treatments are provided in The Pesticide Manual: A WorldCompendium, C. D. S. Tomlin Ed., Published by the British CropProduction Council, which is hereby incorporated by reference.

Some seed treatments that may be used on crop seed include, but are notlimited to, one or more of abscisic acid, acibenzolar-S-methyl,avermectin, amitrol, azaconazole, azospirillum, azadirachtin,azoxystrobin, Bacillus spp. (including one or more of cereus, firmus,megaterium, pumilis, sphaericus, subtilis and/or thuringiensis species),bradyrhizobium spp. (including one or more of betae, canariense,elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and/oryuanmingense), captan, carboxin, chitosan, clothianidin, copper,cyazypyr, difenoconazole, etidiazole, fipronil, fludioxonil,fluoxastrobin, fluquinconazole, flurazole, fluxofenim, harpin protein,imazalil, imidacloprid, ipconazole, isoflavenoids,lipo-chitooligosaccharide, mancozeb, manganese, maneb, mefenoxam,metalaxyl, metconazole, myclobutanil, PCNB, penflufen, penicillium,penthiopyrad, permethrine, picoxystrobin, prothioconazole,pyraclostrobin, rynaxypyr, S-metolachlor, saponin, sedaxane, TCMTB,tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram,tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin,triticonazole and/or zinc. PCNB seed coat refers to EPA RegistrationNumber 00293500419, containing quintozen and terrazole. TCMTB refers to2-(thiocyanomethylthio) benzothiazole.

Seed varieties and seeds with specific transgenic traits may be testedto determine which seed treatment options and application rates maycomplement such varieties and transgenic traits in order to enhanceyield. For example, a variety with good yield potential but head smutsusceptibility may benefit from the use of a seed treatment thatprovides protection against head smut, a variety with good yieldpotential but cyst nematode susceptibility may benefit from the use of aseed treatment that provides protection against cyst nematode, and soon. Likewise, a variety encompassing a transgenic trait conferringinsect resistance may benefit from the second mode of action conferredby the seed treatment, a variety encompassing a transgenic traitconferring herbicide resistance may benefit from a seed treatment with asafener that enhances the plants resistance to that herbicide, etc.Further, the good root establishment and early emergence that resultsfrom the proper use of a seed treatment may result in more efficientnitrogen use, a better ability to withstand drought and an overallincrease in yield potential of a variety or varieties containing acertain trait when combined with a seed treatment.

Methods for Killing an Insect Pest and Controlling an Insect Population

In some embodiments methods are provided for killing an insect pest,comprising contacting the insect pest with an insecticidally-effectiveamount of a recombinant PtIP-83 polypeptide. In some embodiments methodsare provided for killing an insect pest, comprising contacting theinsect pest with an insecticidally-effective amount of a recombinantpesticidal protein of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO:758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO:767, SEQ ID NO: 768, SEQ ID NO: 769 or a variant thereof.

In some embodiments methods are provided for killing an insect pest,comprising contacting the insect pest with an insecticidally-effectiveamount of a recombinant PtIP-83 polypeptide of SEQ ID NO: 236-299, SEQID NO: 334-367, SEQ ID NO: 398-427, SEQ ID NO: 518-607, SEQ ID NO:640-645, and SEQ ID NO: 728-737.

In some embodiments methods are provided for killing an insect pest,comprising contacting the insect pest with an insecticidally-effectiveamount of a recombinant PtIP-83 polypeptide comprising an amino acidsequence of any one of SEQ ID NO: 786-888 or a variant thereof.

In some embodiments methods are provided for controlling an insect pestpopulation, comprising contacting the insect pest population with aninsecticidally-effective amount of a recombinant PtIP-83 polypeptide. Insome embodiments methods are provided for controlling an insect pestpopulation, comprising contacting the insect pest population with aninsecticidally-effective amount of a recombinant PtIP-83 polypeptide ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ IDNO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759,SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ IDNO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768,SEQ ID NO: 769 or a variant thereof. As used herein, “controlling a pestpopulation” or “controls a pest” refers to any effect on a pest thatresults in limiting the damage that the pest causes. Controlling a pestincludes, but is not limited to, killing the pest, inhibitingdevelopment of the pest, altering fertility or growth of the pest insuch a manner that the pest provides less damage to the plant,decreasing the number of offspring produced, producing less fit pests,producing pests more susceptible to predator attack or deterring thepests from eating the plant.

In some embodiments methods are provided for controlling an insect pestpopulation, comprising contacting the insect pest population with aninsecticidally-effective amount of a recombinant PtIP-83 polypeptide ofany one of SEQ ID NO: 236-299, SEQ ID NO: 334-367, SEQ ID NO: 398-427,SEQ ID NO: 518-607, SEQ ID NO: 640-645, and SEQ ID NO: 728-737.

In some embodiments methods are provided for controlling an insect pestpopulation, comprising contacting the insect pest population with aninsecticidally-effective amount of a recombinant PtIP-83 polypeptide ofany one of SEQ ID NO: 786-888 or a variant thereof.

In some embodiments methods are provided for protecting a plant from aninsect pest, comprising expressing in the plant or cell thereof at leastone recombinant polynucleotide encoding a PtIP-83 polypeptide. In someembodiments methods are provided for protecting a plant from an insectpest, comprising expressing in the plant or cell thereof a recombinantpolynucleotide encoding a PtIP-83 polypeptide of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ IDNO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQID NO: 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO:756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID NO:765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768 or SEQ ID NO: 769 orvariants thereof.

In some embodiments methods are provided for protecting a plant from aninsect pest, comprising expressing in the plant or cell thereof at leastone recombinant polynucleotide encoding a PtIP-83 polypeptide. In someembodiments methods are provided for protecting a plant from an insectpest, comprising expressing in the plant or cell thereof a recombinantpolynucleotide encoding a PtIP-83 polypeptide of any one of SEQ ID NO:236-299, SEQ ID NO: 334-367, SEQ ID NO: 398-427, SEQ ID NO: 518-607, SEQID NO: 640-645, and SEQ ID NO: 728-737.

In some embodiments methods are provided for protecting a plant from aninsect pest, comprising expressing in the plant or cell thereof at leastone recombinant polynucleotide encoding a PtIP-83 polypeptide. In someembodiments methods are provided for protecting a plant from an insectpest, comprising expressing in the plant or cell thereof a recombinantpolynucleotide encoding a PtIP-83 polypeptide of any one of SEQ ID NO:786-888 or a variant thereof.

Insect Resistance Management (IRM) Strategies

Expression of B. thuringiensis δ-endotoxins in transgenic corn plantshas proven to be an effective means of controlling agriculturallyimportant insect pests (Perlak, et al., 1990; 1993). However, insectshave evolved that are resistant to B. thuringiensis δ-endotoxinsexpressed in transgenic plants. Such resistance, should it becomewidespread, would clearly limit the commercial value of germplasmcontaining genes encoding such B. thuringiensis b-endotoxins.

One way to increasing the effectiveness of the transgenic insecticidesagainst target pests and contemporaneously reducing the development ofinsecticide-resistant pests is to use provide non-transgenic (i.e.,non-insecticidal protein) refuges (a section of non-insecticidalcrops/corn) for use with transgenic crops producing a singleinsecticidal protein active against target pests. The United StatesEnvironmental Protection Agency(epa.gov/oppbppdl/biopesticides/pips/bt_corn_refuge_2006.htm, which canbe accessed using the www prefix) publishes the requirements for usewith transgenic crops producing a single Bt protein active againsttarget pests. In addition, the National Corn Growers Association, ontheir website:(ncga.com/insect-resistance-management-fact-sheet-bt-corn, which can beaccessed using the www prefix) also provides similar guidance regardingrefuge requirements. Due to losses to insects within the refuge area,larger refuges may reduce overall yield.

Another way of increasing the effectiveness of the transgenicinsecticides against target pests and contemporaneously reducing thedevelopment of insecticide-resistant pests would be to have a repositoryof insecticidal genes that are effective against groups of insect pestsand which manifest their effects through different modes of action.

Expression in a plant of two or more insecticidal compositions toxic tothe same insect species, each insecticide being expressed at efficaciouslevels would be another way to achieve control of the development ofresistance. This is based on the principle that evolution of resistanceagainst two separate modes of action is far more unlikely than only one.Roush, for example, outlines two-toxin strategies, also called“pyramiding” or “stacking,” for management of insecticidal transgeniccrops. (The Royal Society. Phil. Trans. R. Soc. Lond. B. (1998)353:1777-1786). Stacking or pyramiding of two different proteins eacheffective against the target pests and with little or nocross-resistance can allow for use of a smaller refuge. The USEnvironmental Protection Agency requires significantly less (generally5%) structured refuge of non-Bt corn be planted than for single traitproducts (generally 20%). There are various ways of providing the IRMeffects of a refuge, including various geometric planting patterns inthe fields and in-bag seed mixtures, as discussed further by Roush.

In some embodiments the PtIP-83 polypeptide of the disclosure are usefulas an insect resistance management strategy in combination (i.e.,pyramided) with other pesticidal proteins include but are not limited toBt toxins, Xenorhabdus sp. or Photorhabdus sp. insecticidal proteins,and the like.

Provided are methods of controlling Lepidoptera and/or Coleoptera insectinfestation(s) in a transgenic plant that promote insect resistancemanagement, comprising expressing in the plant at least two differentinsecticidal proteins having different modes of action.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management the at least one of the insecticidal proteinscomprise a PtIP-83 polypeptide insecticidal to insects in the orderLepidoptera and/or Coleoptera.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management the at least one of the insecticidal proteinscomprises a PtIP-83 polypeptide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ IDNO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO:757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQID NO: 762, SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO:766, SEQ ID NO: 767, SEQ ID NO: 768, SEQ ID NO: 769 or variants thereof,insecticidal to insects in the order Lepidoptera and/or Coleoptera.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management the at least one of the insecticidal proteinscomprises a PtIP-83 polypeptide of any one of SEQ ID NO: 236-299, SEQ IDNO: 334-367, SEQ ID NO: 398-427, SEQ ID NO: 518-607, SEQ ID NO: 640-645,and SEQ ID NO: 728-737.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management comprise expressing in the transgenic plant aPtIP-83 polypeptide and a Cry protein insecticidal to insects in theorder Lepidoptera and/or Coleoptera having different modes of action.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation of a transgenic plant and promoting insectresistance management comprise in the transgenic plant a PtIP-83polypeptide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ IDNO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758,SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ IDNO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767,SEQ ID NO: 768, SEQ ID NO: 769 or variants thereof and a Cry proteininsecticidal to insects in the order Lepidoptera and/or Coleopterahaving different modes of action.

Also provided are methods of reducing likelihood of emergence ofLepidoptera and/or Coleoptera insect resistance to transgenic plantsexpressing in the plants insecticidal proteins to control the insectspecies, comprising expression of a PtIP-83 polypeptide insecticidal tothe insect species in combination with a second insecticidal protein tothe insect species having different modes of action.

Also provided are means for effective Lepidoptera and/or Coleopterainsect resistance management of transgenic plants, comprisingco-expressing at high levels in the plants two or more insecticidalproteins toxic to Lepidoptera and/or Coleoptera insects but eachexhibiting a different mode of effectuating its killing activity,wherein the two or more insecticidal proteins comprise a PtIP-83polypeptide and a Cry protein. Also provided are means for effectiveLepidoptera and/or Coleoptera insect resistance management of transgenicplants, comprising co-expressing at high levels in the plants two ormore insecticidal proteins toxic to Lepidoptera and/or Coleopterainsects but each exhibiting a different mode of effectuating its killingactivity, wherein the two or more insecticidal proteins comprise aPtIP-83 polypeptide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO:758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO:767, SEQ ID NO: 768, SEQ ID NO: 769 or variants thereof and a Cryprotein.

In addition, methods are provided for obtaining regulatory approval forplanting or commercialization of plants expressing proteins insecticidalto insects in the order Lepidoptera and/or Coleoptera, comprising thestep of referring to, submitting or relying on insect assay binding datashowing that the PtIP-83 polypeptide does not compete with binding sitesfor Cry proteins in such insects. In addition, methods are provided forobtaining regulatory approval for planting or commercialization ofplants expressing proteins insecticidal to insects in the orderLepidoptera and/or Coleoptera, comprising the step of referring to,submitting or relying on insect assay binding data showing that thePtIP-83 polypeptide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO:758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO:767, SEQ ID NO: 768, SEQ ID NO: 769 or variant thereof does not competewith binding sites for Cry proteins in such insects.

Methods for Increasing Plant Yield

Methods for increasing plant yield are provided. The methods compriseproviding a plant or plant cell expressing a polynucleotide encoding thepesticidal polypeptide sequence disclosed herein and growing the plantor a seed thereof in a field infested with a pest against which thepolypeptide has pesticidal activity. In some embodiments, thepolypeptide has pesticidal activity against a Lepidopteran, Coleopteran,Dipteran, Hemipteran or nematode pest, and the field is infested with aLepidopteran, Hemipteran, Coleopteran, Dipteran or nematode pest.

As defined herein, the “yield” of the plant refers to the quality and/orquantity of biomass produced by the plant. “Biomass” as used hereinrefers to any measured plant product. An increase in biomass productionis any improvement in the yield of the measured plant product.Increasing plant yield has several commercial applications. For example,increasing plant leaf biomass may increase the yield of leafy vegetablesfor human or animal consumption. Additionally, increasing leaf biomasscan be used to increase production of plant-derived pharmaceutical orindustrial products. An increase in yield can comprise any statisticallysignificant increase including, but not limited to, at least a 1%increase, at least a 3% increase, at least a 5% increase, at least a 10%increase, at least a 20% increase, at least a 30%, at least a 50%, atleast a 70%, at least a 100% or a greater increase in yield compared toa plant not expressing the pesticidal sequence.

In specific methods, plant yield is increased as a result of improvedpest resistance of a plant expressing a PtIP-83 polypeptide disclosedherein. Expression of the PtIP-83 polypeptide results in a reducedability of a pest to infest or feed on the plant, thus improving plantyield.

Methods of Processing

Further provided are methods of processing a plant, plant part or seedto obtain a food or feed product from a plant, plant part or seedcomprising a PtIP-83 polypeptide. The plants, plant parts or seedsprovided herein, can be processed to yield oil, protein products and/orby-products that are derivatives obtained by processing that havecommercial value. Non-limiting examples include transgenic seedscomprising a nucleic acid molecule encoding a PtIP-83 polypeptide whichcan be processed to yield soy oil, soy products and/or soy by-products.

“Processing” refers to any physical and chemical methods used to obtainany soy product and includes, but is not limited to, heat conditioning,flaking and grinding, extrusion, solvent extraction or aqueous soakingand extraction of whole or partial seeds

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTALS Example 1—Identification of an Insecticidal Protein ActiveAgainst Broad Spectrum of Lepidopteran Insects from the Fern, AdiantumPedatum, (PS-7140)

The insecticidal protein PtIP-83Aa (SEQ ID NO: 1) was identified byprotein purification, mass spectroscopy (MS) and PCR cloning fromAdiantum pedatum, (PS-7140) as follows.

Adiantum pedatum was collected by a collaborator and assignedidentification number PS-7140. PS-7140 was collected, flash frozen inliquid N₂ and stored at −80° C. After storage it was ground to a finepowder at liquid N₂ temperatures with a Geno Ball Mill (SPEX, Metuchen,N.J.). To extract protein, 20 mL of 50 mM Tris buffer, pH 8.0, 150 mMKCl, 2.5 mM EDTA, 1.5% polyvinylpolypyrrolidone (PVPP) and proteaseinhibitor cocktail (Roche Diagnostics, Germany) was added to every 5 gfresh weight of tissue. The homogenate was centrifuged to remove celldebris, filtered through 0.22 um filters and desalted using 10 ml ZebaSpin Desalting columns (Thermo Scientific, IL.)

Bioassays against the three pest species, Soybean Looper (SBL)(Chrysodeixis includens), Corn Earworm (CEW) (Helicoverpa zea) andEuropean Corn Borer (ECB) (Ostrinia nubilalis) were conducted using thedesalted protein extract overlaid onto an agar-based Lepidoptera diet(Southland Products Inc., Lake Village, Ark.) in a 96-well plate format.Six replicates were used per sample. Samples were allowed to dry on topof the diet and two to five neonate insects were placed into each wellof the treated plate. After four days of incubation at 27° C. larvaewere scored for mortality or severity of stunting. The scores wererecorded numerically as dead (3), severely stunted (2) (little or nogrowth but alive and equivalent to a 1^(st) instar larvae), stunted (1)(growth to second instar but not equivalent to controls), or normal (0).Subjecting the sample to proteinase K and heat treatments resulted inloss of activity indicating that the active principle was proteinaceousin nature. Bioassay results are shown in Table 6.

TABLE 6 Activity of A. pedatum crude protein extract against Lepidopteralarvae Ave. Score after Proteinase Ave. Score K/Heat Neonate SoybeanLooper 3 0 Corn Earworm 2 0 European Corn Borer 1.5 0

For protein purification, PS-7140 fronds were ground to a fine powder atliquid N₂ temperatures with a Geno Ball Mill (SPEX, Metuchen, N.J.).Protein was extracted in 100 mM Tris buffer, pH 8.0, 150 mM KCl, 2.5 mMEDTA, 1.5% PVPP and protease inhibitor cocktail (Roche Diagnostics,Germany). The extracted material was centrifuged to remove cell debris,filtered through Miracloth™ (Calbiochem) and then ammonium sulfate addedto 35% and allowed to equilibrate. The suspension was centrifuged andthe resulting pellet was resuspended in a small volume of 20 mM Trisbuffer, pH 8. After clarification by centrifugation it was desaltedusing a Sephadex G25 column (GE, Piscataway, N.J.) equilibrated in 20 mMTris buffer, pH 8. The desalted protein fraction pool was loaded onto a1 ml Mono Q column (GE, Piscataway, N.J.) and eluted with a linear (60CV (column volumes) gradient from 0 M to 0.7 M NaCl in 20 mM Tris, pH8.0 Fractions active against SBL and ECB were combined and desalted into25 mM MOPS, pH 6.7. This was then loaded onto a 4 mL Mono P column(Buffer A: 25 mM MOPS, pH 6.7; Buffer B: Polybuffer 74, pH 4) using a 4CV linear gradient (0% Buffer B) followed by a 15 CV 100% Buffer B wash.1 mL fractions were collected. Fractions 47 and 48 showed activityagainst ECB and SBL. Based on LDS-PAGE these active fractions containeda protein band at approximately 95 kDa. The protein representing the 95kDa band was named PtIP-83Aa (SEQ ID NO: 1).

Protein identification was performed by MS analysis after proteindigestion with trypsin. Proteins for MS identification were obtainedafter running the sample on an LDS-PAGE gel stained with Brilliant BlueG-250 Stain. Bands of interest were excised from the gel, de-stained,reduced with dithiothreitol and then alkylated with iodoacetamide.Following overnight digestion with trypsin, samples were analyzed bynano-liquid chromatography/electrospray tandem mass spectrometry(nano-LC/ES-MSMS) on a Thermo Q Exactive Orbitrap mass spectrometer(Thermo Fisher Scientific) interfaced with an Eksigent NanoLC Ultra 1-DPlus nano-Ic system and a nanolc-as2 autosampler (AB Sciex). The proteinidentification was performed by searching the nano-LC/MSMS data againstan in-house transcriptome database containing the transcripts from thesource plant materials and the public protein database Swiss-Prot usingthe Mascot search engine (Matrix Science).

The amino acid sequence of SEQ ID NO: 1 was BLAST (Basic Local AlignmentSearch Tool; Altschul, et al., (1993) J. Mol. Biol. 215:403-410; seealso ncbi.nlm.nih.gov/BLAST/, which can be accessed using the wwwprefix) searched against public and DUPONT-PIONEER internal databasesthat included plant protein sequences. Amino acid sequences were alignedwith proteins in a proprietary DUPONT-PIONEER plant protein database.

Example 2 Transcriptomic Sequencing of PtIP-83Aa

A transcriptome for Adiantum pedatum, PS-7140 was prepared as follows.Total RNAs were isolated from frozen tissues by use of the Qiagen®RNeasy® kit for total RNA isolation. Sequencing libraries from theresulting total RNAs were prepared using the TruSeq™ mRNA-Seq kit andprotocol from Illumina®, Inc. (San Diego, Calif.). Briefly, mRNAs wereisolated via attachment to oligo(dT) beads, fragmented to a mean size of180 nt, reverse transcribed into cDNA by random hexamer prime, endrepaired, 3′ A-tailed, and ligated with Illumina® indexed TruSeq™adapters. Ligated cDNA fragments were PCR amplified using Illumina®TruSeq™ primers and purified PCR products were checked for quality andquantity on the Agilent Bioanalyzer® DNA 7500 chip. Post quality andquantity assessment, 100 ng of the transcript library was normalized bytreatment with Duplex Specific Nuclease (DSN) (Evrogen®, Moscow,Russia). Normalization was accomplished by addition of 200 mM Hepesbuffer, followed by heat denaturation and five hour anneal at 68° C.Annealed library was treated with 2 ul of DSN enzyme for 25 minutes,purified by Qiagen® MinElute® columns according to manufacturerprotocols, and amplified twelve cycles using Illumina® adapter specificprimers. Final products were purified with Ampure® XP beads (BeckmanGenomics, Danvers, Mass.) and checked for quality and quantity on theAgilent Bioanalyzer® DNA 7500 chip.

Normalized transcript libraries were sequenced according to manufacturerprotocols on the Illumina® Genome Analyzer IIx. Each library washybridized to two flow cell lanes and amplified, blocked, linearized andprimer hybridized using the Illumina clonal cluster generation processon cBot®. Sequencing was completed on the Genome Analyzer IIx,generating sixty million 75 bp paired end reads per normalized library.

Peptide sequence identified for PtIP-83Aa (SEQ ID NO: 1) by LC-MS/MS/MSsequencing (described in Example 1) were searched against the proteinsequences predicted by open reading frames (ORFs) from the internaltranscriptome for PS-7140CF assemblies. The peptides gave a perfectmatch to a transcript corresponding to PtIP-83Aa (SEQ ID NO: 2). Thecoding sequences were used to design the following primers to clone thePtIP-83Aa coding sequence:

(95KD N-T Nco I for) (SEQ ID NO: 30) CCATGGCTCTCGTGGATTACGGCAAG and(95KD C-T Hpa I REV) (SEQ ID NO: 31) GTTAACCTACTCTTCGTCGTGCCGCCAGTC.This clone was produced by polymerase chain reaction using the KOD HotStart DNA Polymerase® PCR kit (Novagen, Merck KGaA, Darmstadt, Germany)and the total RNA from Adiantum pedatum as a template. The cloned PCRproduct was confirmed by sequencing.

Based on the DNA and protein sequencing, the PtIP-83Aa polynucleotidesequence is shown as SEQ ID NO: 2 and the polypeptide sequence as SEQ IDNO: 1.

Example 3—Identification of PtIP-83 Homologs

Gene identities may be determined by conducting BLAST® (Basic LocalAlignment Search Tool; Altschul, et al., (1993) J. Mol. Biol.215:403-410; see also ncbi.nlm.nih.gov/BLAST/, which can be accessedusing the www prefix) searches under default parameters for similarityto sequences. The polynucleotide sequence for PtIP-83Aa (SEQ ID NO: 2)was analyzed. No close homologs of PtIP-83Aa (SEQ ID NO: 1) wereidentified in public databases.

Gene identities conducted by BLAST® in a DUPONT PIONEER internaltranscriptome database of ferns and other primitive plants identifiedhomologs for PtIP-83Aa (SEQ ID NO: 1). The PtIP-83Aa homologs and theorganism they were identified from are shown in Table 7.

TABLE 7 Sequence id no Source Organism PtIP-83Aa SEQ ID NO: 1 PS-7140Adiantum pedatum PtIP-83Ca SEQ ID NO: 5 PS-11481 Adiantum trapeziformevar. braziliense PtIP-83Cb SEQ ID NO: 7 PS-11481 Adiantum trapeziformevar. braziliense PtIP-83Cc SEQ ID NO: 9 PS-11481 Adiantum trapeziformevar. braziliense PtIP-83Cd SEQ ID NO: 11 PS-11481 Adiantum trapeziformevar. braziliense PtIP-83Ce SEQ ID NO: 13 PS-12345 Adiantum peruvianumPtIP-83Cf SEQ ID NO: 15 PS-9224 Lygodium flexuosum PtIP-83Cg SEQ ID NO:17 PS-12345 Adiantum peruvianum PtIP-83Da SEQ ID NO: 19 PS-12345Adiantum peruvianum PtIP-83Ea SEQ ID NO: 21 PS-11481 Adiantumtrapeziforme var. braziliense PtIP-83Eb SEQ ID NO: 23 PS-11481 Adiantumtrapeziforme var. braziliense PtIP-83Fa SEQ ID NO: 3 PS-8568 Microsorummusifolium PtIP-83Fb SEQ ID NO: 716 PS-9319 Polypodium punctatum‘Serratum’ PtIP-83Ch SEQ ID NO: 754 LW13327 Polystichium tsus-simensePtIP-83Ch- SEQ ID NO: 755 LW13327 Polystichium tsus-simense likePtIP-83Fd SEQ ID NO: 756 LW13327 Polystichium tsus-simense PtIP-83Fe SEQID NO: 757 LW13327 Polystichium tsus-simense PtIP-83Cj SEQ ID NO: 758LW12354 Rumohra adiantiformis PtIP-83Cj- SEQ ID NO: 759 LW12354 Rumohraadiantiformis like PtIP-83Ff SEQ ID NO: 760 LW12354 Rumohraadiantiformis PtIP-83Ff- SEQ ID NO: 761 LW12354 Rumohra adiantiformislike PtIP-83Cj SEQ ID NO: 762 NY012 Asplenium trichomanes PtIP-83Cj- SEQID NO: 763 NY012 Asplenium trichomanes like PtIP-83Ga SEQ ID NO: 764NY009 Phyllitis scolopendium ‘Angustifolia’ PtIP-83Ga- SEQ ID NO: 765NY009 Phyllitis scolopendium like ‘Angustifolia’ PtIP-83Fg SEQ ID NO:766 NY009 Phyllitis scolopendium ‘Angustifolia’ PtIP-83Fh SEQ ID NO: 767NY009 Phyllitis scolopendium ‘Angustifolia’ PtIP-83Fi SEQ ID NO: 768NY009 Phyllitis scolopendium ‘Angustifolia’ PtIP-83Fi- SEQ ID NO: 769NY009 Phyllitis scolopendium like ‘Angustifolia’

cDNA was generated from source organisms with identified homologs byreverse transcription from total RNA. Homologs were PCR amplified fromtheir respective cDNAs using primers designed to the coding sequences ofeach homolog and subcloned into a plant transient vector containing theDMMV promoter. Cloned PCR products were confirmed by sequencing. Cloningprimers are shown in Table 8. The cDNA for homolog PtIP-83Fb wassynthesized (SEQ ID NO: 717) based on the transcriptome assembly ofPS-9319 and subcloned into a plant transient vector.

TABLE 8 Gene Primer Sequence PtIP-83Cb GZ-550-CGAAATCTCTCATCTAAGAGGCTGGATCCTAGGATGGATTACAGCAC 83Ca-FGCTTTACAGGGAC (SEQ ID NO: 608) PtIP-83Cb GZ-550-TTAAGTTGGCCAATCCAGAAGATGGACAAGTCTAGACTACTCCTCCT 83Ca-RCTTGCCGCCAGTC (SEQ ID NO: 609) PtIP-83Ca, Cc, and GZ-550-CGAAATCTCTCATCTAAGAGGCTGGATCCTAGGATGGATTACAGCAC Cd 83Ca-FGCTTTACAGGGAC (SEQ ID NO: 608) PtIP-83Ca, Cc, and 83Ca-1s-RCAA GGA TTG CAT TGC TAG GAA GG (SEQ ID NO: 611) Cd PtIP-83Ca, Cc, andGZ-550- TTAAGTTGGCCAATCCAGAAGATGGACAAGTCTAGACTACTCCTCCT Cd 83Ca-RCTTGCCGCCAGTC (SEQ ID NO: 609) PtIP-83Ca, Cc, and 83Ca-1sCCTTCCTAGCAATGCAATCCTTG (SEQ ID NO: 613) Cd PtIP-83Ce GZ-550-CGAAATCTCTCATCTAAGAGGCTGGATCCTAGGATGGATTACAGCAC 83Ca-FGCTTTACAGGGAC (SEQ ID NO: 608) PtIP-83Ce 550-AAGTTGGCCAATCCAGAAGATGGACAAGTCTAGACTACTCCTCCTCT 12345-RTTCTCCTCCTGCC (SEQ ID NO: 615) PtIP-83Cf 550-9224-CGAAATCTCTCATCTAAGAGGCTGGATCCTAGGATGGCCAGTGTACT F1GGATTACAGCAC (SEQ ID NO: 616) PtIP-83Cf 550-9224-TTAAGTTGGCCAATCCAGAAGATGGACAAGTCTAGACTACTCCTCCT R1CGTGCCGCC (SEQ ID NO: 617) PtIP-83Cg 550-GAAATCTCTCATCTAAGAGGCTGGATCCTAGGATGGATTACAGCACT 11790.2-FCTTTACAGGGATC (SEQ ID NO: 618) PtIP-83Cg GZ-550-TTAAGTTGGCCAATCCAGAAGATGGACAAGTCTAGACTACTCCTCCT 83Ca-RCTTGCCGCCAGTC (SEQ ID NO: 609) PtIP-83Da GZ-550-CGAAATCTCTCATCTAAGAGGCTGGATCCTAGGATGGATTACAGCAC 83Ca-FGCTTTACAGGGAC (SEQ ID NO: 608) PtIP-83Da 550-GTTGGCCAATCCAGAAGATGGACAAGTCTAGATTAGAGTGGCTTCGC UTR8.7-RCAGTGTCG (SEQ ID NO: 621) PtIP-83Ea GZ-550-CGAAATCTCTCATCTAAGAGGCTGGATCCTAGGATGGATTACAGCAC 83Ca-FGCTTTACAGGGAC (SEQ ID NO: 608) PtIP-83Ea 550-AAGTTGGCCAATCCAGAAGATGGACAAGTCTAGACTACTCCTCCTCT 12345-RTTCTCCTCCTGCC (SEQ ID NO: 615) PtIP-83Eb GZ-550-CGAAATCTCTCATCTAAGAGGCTGGATCCTAGGATGGATTACAGCAC 83Ca-FGCTTTACAGGGAC (SEQ ID NO: 608) PtIP-83Eb 550-AAGTTGGCCAATCCAGAAGATGGACAAGTCTAGACTACTCCTCCTCT 12345-RTTCTCCTCCTGCC (SEQ ID NO: 615) PtIP-83Fa infusionTTTAACTTAGCCTAGGATCCATGGAATATAGCAGCTTGTAC bamHI (SEQ ID NO: 32)PtIP-83Fa infusion ACTCCTTCTTTAGTTAACTTACTCCACATCACCCTCTTGTCG HpaI(SEQ ID NO: 33) PtIP-83Ch and LW13327-cgaaatctctcatctaagaggctggatcctaggATGGATTACAGCACGCTTTACA PtIP-83Ch-likeF1 GG (SEQ ID NO: 770) PtIP-83Ch and LW13327-taagttggccaatccagaagatggacaagtctagaCTACTCCTCCACCTCCTGCCT PtIP-83Ch-likeR1 CC (SEQ ID NO: 771) PtIP-83Fd and LW13327-cgaaatctctcatctaagaggctggatcctaggATGACGATGGCGGCAACTG PtIP-83Fe F2(SEQ ID NO: 772) PtIP-83Fd and LW13327-ggccaatccagaagatggacaagtctagaCTAGAAAGAAATTTTCCTGATAGC PtIP-83Fe R2TGAG (SEQ ID NO: 773) PtIP-83Ci and PtIP- LW12354-cgaaatctctcatctaagaggctggatcctaggATGGATTACAGCACTCTTTACA 83Ci-like F1CGG (SEQ ID NO: 774) PtIP-83Ci and PtIP- LW12354-taagttggccaatccagaagatggacaagtctagaCTACTCCTCTTGCCGCCAGT 83Ci-like R1C (SEQ ID NO: 775) PtIP-83Ff and PtIP- LW12354-cgaaatctctcatctaagaggctggatcctaggATGGCTGCCTCCGCTGCTG 83Ff-like F2(SEQ ID NO: 776) PtIP-83Ff and PtIP- LW12354-taagttggccaatccagaagatggacaagtctagaCTAGAAAGAAATGCGCCGG 83Ff-like R2ATAG (SEQ ID NO: 777) PtIP-83Cj and PtIP- NY012-F1cgaaatctctcatctaagaggctggatcctaggATGGATTACAGCACGCTTTACA 83Cj-likeGG (SEQ ID NO: 778) PtIP-83Cj and PtIP- NY012-R1taagttggccaatccagaagatggacaagtctagaCTACTCCTCCTCCTCCTCGTG 83Cj-likeCC (SEQ ID NO: 779) PtIP-83Ga and NY009-F1cgaaatctctcatctaagaggctggatcctaggATGGGTGTCACAGTCGTTAGC PtIP-83Ga-likeG (SEQ ID NO: 780) PtIP-83Ga and NY009-R1taagttggccaatccagaagatggacaagtctagaTTAGCTGACGACCTGATCAT PtIP-83Ga-likeCGC (SEQ ID NO: 781) PtIP-83Fg, Fi, and NY009-F2cgaaatctctcatctaagaggctggatcctaggATGGAGTATGGCAGCTTGTAT Fi-likeGG (SEQ ID NO: 782) PtIP-83Fg, Fi, and NY009-gttggccaatccagaagatggacaagtctagaCTATAACTCCGCATCAGCTCGTT Fi-like R2aG (SEQ ID NO: 783) PtIP-83Fh NY009-F3cgaaatctctcatctaagaggctggatcctaggATGGAGTACTCCGACTTGTATGAGG (SEQ ID NO: 784) PtIP-83Fh NY009-R3taagttggccaatccagaagatggacaagtctagaTCACTCCTCATCGACTTCCCG (SEQ ID NO: 785)

Additional diversity was recovered by PCR amplification with primersdesigned to the non-coding regions flanking identified homologs. Primerswere designed to conserved sequences in the 5′ and 3′ untranslatedregions of PtIP-83Ca BLAST hits within the DUPONT PIONEER internaltranscriptome database. Primers pairs (1) GCCTTTATCGACTCCTAATTCACACC(SEQ ID NO: 626) and CCACATTGTGCATTACGACCAC (SEQ ID NO: 627), and (2)CCAGTGATTTGAGTTCCTTCATTATG (SEQ ID NO: 628) and GAACAGTACATTGACTGCATGTGC(SEQ ID NO: 629) were used to generate PCR products from PS-11481 andPS-12345 cDNA. Resulting PCR products were blunt end cloned using theZero Blunt® TOPO® PCR Cloning Kit (Invitrogen) and sequence analyzed.From this analysis, homologs PtIP-83Cg, Da, Ea, and Eb were identifiedand subcloned into the plant transient vector. Cloning primers are shownin Table 8.

The amino acid sequence identity of the PtIP-83Aa homologs as calculatedusing the Needleman-Wunsch algorithm, as implemented in the Needleprogram (EMBOSS tool suite) are shown in Table 9a-9c.

TABLE 9a PtIP- PtIP- PtIP- PtIP- PtIP- PtIP- PtIP- PtIP- PtIP- PtIP-83Ca 83Cb 83Cc 83Cd 83Ce 83Cf 83Cg 83Ch-like 83Ch 83Ci-like SEQ ID SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 5 NO: 7NO: 9 NO: 11 NO: 13 NO: 15 NO: 17 NO: 755 NO: 754 NO: 759 PtIP-83Aa 71.471.2 71.9 72.0 71.5 77.4 71.5 74.8 74.8 70.9 SEQ ID NO: 1 PtIP-83Ca —76.4 98.5 98.2 98.0 79.3 76.6 80.6 80.6 75.8 SEQ ID NO: 5 PtIP-83Cb — —76.7 76.8 76.3 77.6 98.6 78.0 78.0 96.6 SEQ ID NO: 7 PtIP-83Cc — — —99.8 99.5 79.1 76.9 80.8 80.8 75.8 SEQ ID NO: 9 PtIP-83Cd — — — — 99.379.3 76.9 80.9 80.9 75.8 SEQ ID NO: 11 PtIP-83Ce — — — — — 78.7 76.580.6 80.6 75.6 SEQ ID NO: 13 PtIP-83Cf — — — — — — 77.8 84.0 84.0 77.3SEQ ID NO: 15 PtIP-83Cg — — — — — — — 78.2 78.2 97.1 SEQ ID NO: 17PtIP-83Ch-like — — — — — — — — 99.9 77.4 SEQ ID NO: 755 PtIP-83Ch — — —— — — — — — 77.4 SEQ ID NO: 754 PtIP-83Ci-like — — — — — — — — — — SEQID NO: 759 PtIP-83Ci — — — — — — — — — — SEQ ID NO: 758 PtIP-83Cj-like —— — — — — — — — — SEQ ID NO: 763 PtIP-83Cj — — — — — — — — — — SEQ IDNO: 762 PtIP-83Da — — — — — — — — — — SEQ ID NO: 19 PtIP-83Ea — — — — —— — — — — SEQ ID NO: 21 PtIP-83Eb — — — — — — — — — — SEQ ID NO: 23PtIP-83Fa — — — — — — — — — — SEQ ID NO: 3 PtIP-83Fb — — — — — — — — — —SEQ ID NO: 716 PtIP-83Fd — — — — — — — — — — SEQ ID NO: 756 PtIP-83Fe —— — — — — — — — — SEQ ID NO: 757 PtIP-83Ff-like — — — — — — — — — — SEQID NO: 761 PtIP-83Ff — — — — — — — — — — SEQ ID NO: 760 PtIP-83Fg — — —— — — — — — — SEQ ID NO: 766 PtIP-83Fh — — — — — — — — — — SEQ ID NO:767 PtIP-83Fi-like — — — — — — — — — — SEQ ID NO: 769 PtIP-83Fi — — — —— — — — — — SEQ ID NO: 768 PtIP-83Ga-like — — — — — — — — — — SEQ ID NO:765

TABLE 9b PtIP- PtIP- PtIP- PtIP- PtIP- PtIP- PtIP- PtIP- PtIP- 83Ci83Cj-like 83Cj 83Da 83Ea 83Eb 83Fa 83Fb 83Fd SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 758 NO: 763 NO: 762 NO: 19 NO: 21NO: 23 NO: 3 NO: 716 NO: 756 PtIP-83Aa 71.0 75.5 73.6 64.7 55.8 55.449.8 50.1 50.8 SEQ ID NO: 1 PtIP-83Ca 75.9 79.5 77.5 89.7 68.6 79.1 48.848.3 48.8 SEQ ID NO: 5 PtIP-83Cb 96.7 79.5 77.2 68.8 59.3 60.2 49.7 49.350.4 SEQ ID NO: 7 PtIP-83Cc 76.0 79.8 77.8 91.1 70.1 80.4 49.2 48.7 49.1SEQ ID NO: 9 PtIP-83Cd 76.0 79.9 77.9 90.8 70.1 80.2 49.2 48.7 49.2 SEQID NO: 11 PtIP-83Ce 75.7 79.8 77.8 90.7 70.5 80.9 49.3 48.8 48.9 SEQ IDNO: 13 PtIP-83Cf 77.4 87.1 84.8 71.4 60.5 61.8 49.3 48.9 52.8 SEQ ID NO:15 PtIP-83Cg 97.2 79.6 77.4 69.1 59.3 60.4 49.5 49.4 50.5 SEQ ID NO: 17PtIP-83Ch-like 77.6 84.9 82.6 72.5 62.5 62.9 50.6 50.6 52.5 SEQ ID NO:755 PtIP-83Ch 77.6 84.9 82.6 72.5 62.5 62.9 50.6 50.7 52.5 SEQ ID NO:754 PtIP-83Ci-like 99.9 79.0 76.8 68.1 58.9 59.9 49.9 49.8 49.3 SEQ IDNO: 759 PtIP-83Ci — 79.1 76.9 68.2 59.1 59.9 50.1 49.9 49.4 SEQ ID NO:758 PtIP-83Cj-like — — 97.0 71.7 61.1 62.7 50.7 50.7 51.5 SEQ ID NO: 763PtIP-83Cj — — — 69.7 61.6 60.7 50.3 50.3 50.4 SEQ ID NO: 762 PtIP-83Da —— — — 61.4 71.7 43.5 43.0 44.4 SEQ ID NO: 19 PtIP-83Ea — — — — — 51.638.7 38.6 38.7 SEQ ID NO: 21 PtIP-83Eb — — — — — — 38.8 38.2 37.9 SEQ IDNO: 23 PtIP-83Fa — — — — — — — 97.1 48.4 SEQ ID NO: 3 PtIP-83Fb — — — —— — — — 48.7 SEQ ID NO: 716 PtIP-83Fd — — — — — — — — — SEQ ID NO: 756PtIP-83Fe — — — — — — — — — SEQ ID NO: 757 PtIP-83Ff-like — — — — — — —— — SEQ ID NO: 761 PtIP-83Ff — — — — — — — — — SEQ ID NO: 760 PtIP-83Fg— — — — — — — — — SEQ ID NO: 766 PtIP-83Fh — — — — — — — — — SEQ ID NO:767 PtIP-83Fi-like — — — — — — — — — SEQ ID NO: 769 PtIP-83Fi — — — — —— — — — SEQ ID NO: 768 PtIP-83Ga-like — — — — — — — — — SEQ ID NO: 765

TABLE 9c PtIP- PtIP- PtIP- PtIP- PtIP- PtIP- PtIP- PtIP- PtIP- 83Fe83Ff-like 83Ff 83Fg 83Fh 83Fi-like 83Fi 83Ga-like 83Ga SEQ ID SEQ ID SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 757 NO: 761 NO: 760 NO:766 NO: 767 NO: 769 NO: 768 NO: 765 NO: 764 PtIP-83Aa 51.0 51.1 50.849.8 47.3 50.2 50.2 34.5 34.6 SEQ ID NO: 1 PtIP-83Ca 49.0 49.2 49.2 47.845.6 47.4 47.4 35.0 35.1 SEQ ID NO: 5 PtIP-83Cb 50.5 51.0 51.0 48.0 47.347.4 47.4 34.5 34.6 SEQ ID NO: 7 PtIP-83Cc 49.2 49.7 49.7 48.1 46.0 47.647.6 35.0 35.1 SEQ ID NO: 9 PtIP-83Cd 49.3 49.8 49.8 48.2 46.1 47.7 47.735.1 35.2 SEQ ID NO: 11 PtIP-83Ce 49.0 49.5 49.5 48.2 46.0 47.7 47.735.0 35.1 SEQ ID NO: 13 PtIP-83Cf 52.5 51.5 51.5 49.7 48.3 49.3 49.335.5 35.6 SEQ ID NO: 15 PtIP-83Cg 50.7 50.4 50.4 47.8 46.9 47.2 47.234.5 34.6 SEQ ID NO: 17 PtIP-83Ch-like 52.4 51.5 51.6 49.7 48.4 49.449.4 34.6 34.7 SEQ ID NO: 755 PtIP-83Ch 52.4 51.5 51.6 49.7 48.1 49.749.7 34.7 34.8 SEQ ID NO: 754 PtIP-83Ci-like 49.9 50.9 50.5 47.9 47.047.6 47.6 34.6 34.7 SEQ ID NO: 759 PtIP-83Ci 50.1 51.0 50.7 48.0 47.147.7 47.7 34.5 34.6 SEQ ID NO: 758 PtIP-83Cj-like 51.5 51.5 51.5 49.346.7 49.2 49.2 35.2 35.3 SEQ ID NO: 763 PtIP-83Cj 50.4 50.2 50.2 48.946.1 48.8 48.8 34.8 34.9 SEQ ID NO: 762 PtIP-83Da 44.6 44.6 44.6 42.540.5 42.0 42.0 30.5 30.6 SEQ ID NO: 19 PtIP-83Ea 38.6 38.7 38.7 39.636.9 39.3 39.3 29.0 29.1 SEQ ID NO: 21 PtIP-83Eb 38.1 38.7 38.7 36.636.1 36.4 36.4 27.4 27.5 SEQ ID NO: 23 PtIP-83Fa 48.3 48.9 49.5 73.464.6 71.9 71.9 31.8 31.9 SEQ ID NO: 3 PtIP-83Fb 48.8 48.9 48.9 73.0 64.371.8 71.8 31.9 32.0 SEQ ID NO: 716 PtIP-83Fd 96.5 72.9 73.0 49.9 47.749.2 49.3 33.8 33.7 SEQ ID NO: 756 PtIP-83Fe — 72.9 73.0 50.0 47.7 49.249.3 33.5 33.4 SEQ ID NO: 757 PtIP-83Ff-like — — 99.9 49.1 46.5 48.448.5 33.1 33.1 SEQ ID NO: 761 PtIP-83Ff — — — 49.1 46.5 48.4 48.5 33.233.2 SEQ ID NO: 760 PtIP-83Fg — — — — 66.4 94.8 94.9 31.8 31.9 SEQ IDNO: 766 PtIP-83Fh — — — — — 65.3 65.4 31.1 31.1 SEQ ID NO: 767PtIP-83Fi-like — — — — — — 99.9 32.1 32.2 SEQ ID NO: 769 PtIP-83Fi — — —— — — — 32.2 32.3 SEQ ID NO: 768 PtIP-83Ga-like — — — — — — — — 99.9 SEQID NO: 765

Example 4: Transient Expression in Leaves and Insect Bioassay

To confirm activity of PtIP-83Aa (SEQ ID NO: 1) a transient expressionsystem under control of a viral promoter pDMMV and/or AtUBQ10 (Dav, et.al., (1999) Plant Mol. Biol. 40:771-782; Norris S R et al (1993) PlantMol Biol. 21(5):895-906) was utilized. The agro-infiltration method ofintroducing an Agrobacterium cell suspension to plant cells of intacttissues so that reproducible infection and subsequent plant derivedtransgene expression may be measured or studied is well known in the art(Kapila, et. al., (1997) Plant Science 122:101-108). Briefly, theunifoliate stage of bush bean (common bean, Phaseolus vulgaris) orsoybean (Glycine max), were agro-infiltrated with normalized bacterialcell cultures of test and control strains. After 4 to 7 days leaf diskswere excised from each plantlet and infested with 2 neonates of SoybeanLooper (SBL) (Chrysodeixis includens), 2 neonates of Corn Earworm (CEW)(Helicoverpa zea), 2 neonates of Fall Armyworm (Spodoptera frugiperda)or 4 neonates of European Corn Borer (ECB) (Ostrinia nubilalis) alone.Control leaf discs were generated with Agrobacterium containing only aDsRed2 fluorescence marker (Clontech™, 1290 Terra Bella Ave. MountainView, Calif. 94043) expression vector. Leaf discs from non-infiltratedplants were included as a second control. The consumption of green leaftissue was scored two (CEW, FAW) or three (ECB, SBL, FAW) days afterinfestation and given scores of 0 to 9. The transiently expressedPtIP-83Aa (SEQ ID NO: 1), protected leaf discs from consumption by theinfested insects while total green tissue consumption was observed forthe negative control and untreated tissue (Table 10). Transient proteinexpression of PtIP-83Aa (SEQ ID NO: 1) was confirmed by massspectrometry-based protein identification using trypsinized proteinextracts of infiltrated leaf tissues (Patterson, (1998) 10(22):1-24,Current Protocol in Molecular Biology published by John Wiley & SonInc). Transient expression can be verified by using HA or EPEA taggedPtIP-83 polypeptides by western analysis of the HA or EPEA tags.

TABLE 10 Transient Leaf Disc Consumption (Scale 1 to 9) expression FAWCEW SBL ECB PtIP-83Aa 8.3 8.3 8.6 8.9 Value Description 1 leaf disk isgreater than 90% consumed 2 leaf disk is 70- 80% consumed 3 leaf disk is60-70% consumed 4 leaf disk is 50-60% consumed 5 leaf disk is 40-50%consumed 6 leaf disk is less than 30% consumed 7 leaf disk is less than10% consumed 8 leaf disk has only a few pinholes 9 leaf disk isuntouched by the insect

Activity of PtIP-83Fa (SEQ ID NO: 4) was validated using the bush beantransient expression system and found to be comparable to PtIP-83Aa (SEQID NO: 2). PtIP-83Fa (SEQ ID NO: 4) showed a similar insecticidalactivity spectrum except for lacking activity against FAW (Table 11).

TABLE 11 Transient Leaf Disc Consumption (Scale 1 to 9) experiment FAWCEW SBL ECB PtIP-83Fa 1.0 6.8 8.1 7.4 Vector control 1.7 2.3 1.1 1.8blank 1.0 1.1 1.4 1.0

Activity of PtIP-83Ca (SEQ ID NO: 5), PtIP-83Cb (SEQ ID NO: 7),PtIP-83Cc (SEQ ID NO: 9), PtIP-83Cd (SEQ ID NO: 11), PtIP-83Ce (SEQ IDNO: 13), PtIP-83Cf (SEQ ID NO: 15), PtIP-83Cg (SEQ ID NO: 17), PtIP-83Da (SEQ ID NO: 19), PtIP-83Ea (SEQ ID NO: 21), PtIP-83Eb (SEQ ID NO:23), PtIP-83Fb (SEQ ID NO: 716), PtIP-83Ch (SEQ ID NO: 754),PtIP-83Ch-like (SEQ ID NO: 755), PtIP-83Fd (SEQ ID NO: 756), PtIP-83Fe(SEQ ID NO: 757), PtIP-83Ci (SEQ ID NO: 758), PtIP-83Ci-like (SEQ ID NO:759), PtIP-83Ff (SEQ ID NO: 760), and PtIP-83Ff-like (SEQ ID NO: 761)were also validated using a bush bean transient expression system. Theactivity spectra for all PtIP-83 homologs are summarized in Table 12,where a “+” indicates an average activity score of <=60% of leaf discconsumed, and a “−” indicates an average activity score of >=60% leafdisc consumed.

TABLE 12 FAW CEW SBL ECB FAW CEW SBL ECB PtIP-83Aa + + + + PtIP-83Ch − −− + PtIP-83Ca − + + + PtIP-83Ch-like − − − + PtIP-83Cb + + + − PtIP-83Fd− − + − PtIP-83Cc + + + + PtIP-83Fe − − − − PtIP-83Cd + + + +PtIP-83Ci + + + + PtIP-83Ce − + + + PtIP-83Ci-like + + + + PtIP-83Cf −− + − PtIP-83Ff − − − − PtIP-83Cg + + + + PtIP-83Ff-like + − + +PtIP-83Da − − − − PtIP-83Cj not tested PtIP-83Ea − − − + PtIP-83Cj-likenot tested PtIP-83Eb − − − − PtIP-83Ga not tested PtIP-83Fa − + + +PtIP-83Ga-like not tested PtIP-83Fb + + + PtIP-83Fg not tested PtIP-83Fhnot tested PtIP-83Fi not tested PtIP-83Fi-like not tested

Example 5: Baculovirus Expression of PtIP-83Aa Polypeptides

The gene encoding PtIP-83Aa SEQ ID NO: 2 was subcloned into thepFastBac™ Dual vector (Invitrogen®) with the stop codon removed forC-terminal translation of a 10×-histidine tag addition (SEQ ID NO: 27)and the sequence of the histidine-tagged PtIP-83Aa polypeptide is setforth as SEQ ID NO: 2. This vector was transformed into DH10Bac cells togenerate baculovirus. These baculovirus were used to infect sf9 insectcells and incubated for 72 hours at 27° C. The infected insect cellswere harvested by centrifugation. The cell culture pellet was suspendedwith 100 mL of lysis buffer (1×PBS, 10% glycerol, with proteaseinhibitor and benzonase) and incubated at 4° C. for 5 min with stirring,then homogenizing twice. The lysate was centrifuged at 16000 rpm for 20min. The supernatant was saved and loaded onto two 2 mL Ni-NTA Hi-BindResin (Novagen, cat #70666) columns pre-equilibrated with Elute buffer(1×PBS, 10% glycerol). The columns were then sequentially eluted with 10mLof Elute buffer containing 10, 20, 50, and 250 mM of imidazole.Samples were analyzed by SDS-PAGE. The purified fractions (E250) wereconcentrated using 100K Amicon® Ultra Centrifugal Filters (Millipore) to˜0.5 mg/mL and demonstrated insecticidal activity against CEW, ECB, FAWand SBL similar to the activity spectrum obtained using transientexpressing leaf discs.

Example 6: Agrobacterium-Mediated Transformation of Maize andRegeneration of Transgenic Plants

For Agrobacterium-mediated transformation of maize with PtIP-83nucleotide sequences such as PtIP-83Aa (SEQ ID NO: 2), PtIP-83Aa ModA(codon optimized) (SEQ ID NO: 28), and PtIP-83Aa ModB (codon optimized(SEQ ID NO: 29), the method of Zhao can be used (U.S. Pat. No. 5,981,840and PCT Patent Publication Number WO 1998/32326; the contents of whichare hereby incorporated by reference). Briefly, immature embryos wereisolated from maize and the embryos contacted with a suspension ofAgrobacterium under conditions whereby the bacteria are capable oftransferring the nucleotide sequence (PtIP-83Aa SEQ ID NO: 2, PtIP-83AaModA (codon optimized)—SEQ ID NO: 28, and PtIP-83Aa ModB (codonoptimized—SEQ ID NO: 29) to at least one cell of at least one of theimmature embryos (step 1: the infection step). In this step the immatureembryos can be immersed in an Agrobacterium suspension for theinitiation of inoculation. The embryos were co-cultured for a time withthe Agrobacterium (step 2: the co-cultivation step). The immatureembryos can be cultured on solid medium following the infection step.Following this co-cultivation period an optional “resting” step iscontemplated. In this resting step, the embryos were incubated in thepresence of at least one antibiotic known to inhibit the growth ofAgrobacterium without the addition of a selective agent for planttransformation (step 3: resting step). The immature embryos werecultured on solid medium with antibiotic, but without a selecting agent,for elimination of Agrobacterium and for a resting phase for theinfected cells. Next, inoculated embryos were cultured on mediumcontaining a selective agent and growing transformed callus is recovered(step 4: the selection step). The immature embryos were cultured onsolid medium with a selective agent resulting in the selective growth oftransformed cells. The callus was then regenerated into plants (step 5:the regeneration step), and calli grown on selective medium werecultured on solid medium to regenerate the plants.

Example 7: Transformation and Regeneration of Soybean (Glycine max)

Transgenic soybean lines are generated by the method of particle gunbombardment (Klein et al., Nature (London) 327:70-73 (1987); U.S. Pat.No. 4,945,050) using a BIORAD Biolistic PDS1000/He instrument and eitherplasmid or fragment DNA. The following stock solutions and media areused for transformation and regeneration of soybean plants:

Stock Solutions:

Sulfate 100× Stock:

37.0 g MgSO₄.7H₂O, 1.69 g MnSO₄.H₂O, 0.86 g ZnSO₄.7H₂O, 0.0025 gCuSO₄.5H₂O

Halides 100× Stock:

30.0 g CaCl₂.2H₂O, 0.083 g KI, 0.0025 g CoCl₂.6H₂O

P, B, Mo 100× Stock:

18.5 g KH₂PO₄, 0.62 g H₃BO₃, 0.025 g Na₂MoO₄.2H₂O

Fe EDTA 100× Stock:

3.724 g Na₂EDTA, 2.784 g FeSO₄.7H₂O

2,4-D Stock:

10 mg/mL Vitamin

B5 vitamins, 1000× Stock:

100.0 g myo-inositol, 1.0 g nicotinic acid, 1.0 g pyridoxine HCl, 10 gthiamine.HCL.

Media (Per Liter):

SB199 Solid Medium:

1 package MS salts (Gibco/BRL—Cat. No. 11117-066), 1 mL B5 vitamins1000× stock, 30 g Sucrose, 4 ml 2, 4-D (40 mg/L final concentration), pH7.0, 2 gm Gelrite

SB1 Solid Medium:

1 package MS salts (Gibco/BRL—Cat. No. 11117-066), 1 mL B5 vitamins1000× stock, 31.5 g Glucose, 2 mL 2, 4-D (20 mg/L final concentration),pH 5.7, 8 g TC agar

SB196:

10 mL of each of the above stock solutions 1-4, 1 mL B5 Vitamin stock,0.463 g (NH4)2 SO4, 2.83 g KNO3, 1 mL 2,4 D stock, 1 g asparagine, 10 gSucrose, pH 5.7

SB71-4:

Gamborg's B5 salts, 20 g sucrose, 5 g TC agar, pH 5.7.

SB103:

1 pk. Murashige & Skoog salts mixture, 1 mL B5 Vitamin stock, 750 mgMgCl2 hexahydrate, 60 g maltose, 2 g Gelrite™, pH 5.7.

SB166:

SB103 supplemented with 5 g per liter activated charcoal.

Soybean Embryogenic Suspension Culture Initiation:

Pods with immature seeds from available soybean plants 45-55 days afterplanting are picked, removed from their shells and placed into asterilized magenta box. The soybean seeds are sterilized by shaking themfor 15 min in a 5% Clorox® solution with 1 drop of Ivory™ soap (i.e., 95mL of autoclaved distilled water plus 5 mL Clorox® and 1 drop of soap,mixed well). Seeds are rinsed using 2 L sterile distilled water andthose less than 3 mm are placed on individual microscope slides. Thesmall end of the seed is cut and the cotyledons pressed out of the seedcoat. Cotyledons are transferred to plates containing SB199 medium(25-30 cotyledons per plate) for 2 weeks, then transferred to SB1 for2-4 weeks. Plates are wrapped with fiber tape. After this time,secondary embryos are cut and placed into SB196 liquid medium for 7days.

Culture Conditions:

Soy bean embryogenic suspension cultures (cv. 93Y21) were maintained in50 mL liquid medium SB196 on a rotary shaker, 100-150 rpm, 26° C. on16:8 h day/night photoperiod at light intensity of 80-100 μE/m2/s.Cultures are subcultured every 7-14 days by inoculating up to ½ dimesize quantity of tissue (clumps bulked together) into 50 mL of freshliquid SB196.

Preparation of DNA for Bombardment:

In particle gun bombardment procedures it is possible to use purified 1)entire plasmid DNA; or 2) DNA fragments containing only the recombinantDNA expression cassette(s) of interest. For every seventeen bombardmenttransformations, 85 μL of suspension is prepared containing 1 to 90picograms (pg) of plasmid DNA per base pair of each DNA plasmid. DNAplasmids or fragments are co-precipitated onto gold particles asfollows. The DNAs in suspension are added to 50 μL of a 10-60 mg/mL 0.6μm gold particle suspension and then combined with 50 μL CaCl₂ (2.5 M)and 20 μL spermidine (0.1 M). The mixture is vortexed for 5 sec, spun ina microfuge for 5 sec, and the supernatant removed. The DNA-coatedparticles are then washed once with 150 μL of 100% ethanol, vortexed andspun in a microfuge again, then resuspended in 85 μL of anhydrousethanol. Five μL of the DNA-coated gold particles are then loaded oneach macrocarrier disc.

Tissue Preparation and Bombardment with DNA:

Approximately 100 mg of two-week-old suspension culture is placed in anempty 60 mm×15 mm petri plate and the residual liquid removed from thetissue using a pipette. The tissue is placed about 3.5 inches away fromthe retaining screen and each plate of tissue is bombarded once.Membrane rupture pressure is set at 650 psi and the chamber is evacuatedto −28 inches of Hg. Following bombardment, the tissue from each plateis divided between two flasks, placed back into liquid media, andcultured as described above.

Selection of Transformed Embryos and Plant Regeneration:

After bombardment, tissue from each bombarded plate is divided andplaced into two flasks of SB196 liquid culture maintenance medium perplate of bombarded tissue. Seven days post bombardment, the liquidmedium in each flask is replaced with fresh SB196 culture maintenancemedium supplemented with 100 ng/ml selective agent (selection medium).For selection of transformed soybean cells the selective agent used canbe a sulfonylurea (SU) compound with the chemical name,2-chloro-N-((4-methoxy-6 methy-1,3,5-triazine-2-yl)aminocarbonyl)benzenesulfonamide (common names: DPX-W4189 and chlorsulfuron).Chlorsulfuron is the active ingredient in the DuPont sulfonylureaherbicide, GLEAN®. The selection medium containing SU is replaced everytwo weeks for 8 weeks. After the 8 week selection period, islands ofgreen, transformed tissue are observed growing from untransformed,necrotic embryogenic clusters. These putative transgenic events areisolated and kept in SB196 liquid medium with SU at 100 ng/ml foranother 5 weeks with media changes every 1-2 weeks to generate new,clonally propagated, transformed embryogenic suspension cultures.Embryos spend a total of around 13 weeks in contact with SU. Suspensioncultures are subcultured and maintained as clusters of immature embryosand also regenerated into whole plants by maturation and germination ofindividual somatic embryos.

Somatic embryos became suitable for germination after four weeks onmaturation medium (1 week on SB166 followed by 3 weeks on SB103). Theyare then removed from the maturation medium and dried in empty petridishes for up to seven days. The dried embryos are then planted inSB71-4 medium where they are allowed to germinate under the same lightand temperature conditions as described above. Germinated embryos aretransferred to potting medium and grown to maturity for seed production.

Example 8—Particle Bombardment Transformation and Regeneration ofTransgenic Plants

Immature maize embryos from greenhouse donor plants are bombarded with aplasmid containing a nucleotide sequence encoding the insecticidalprotein. The ears are husked and surface sterilized in 30% Clorox®bleach plus 0.5% Micro detergent for 20 minutes and rinsed two timeswith sterile water. The immature embryos are excised and placed embryoaxis side down (scutellum side up), 25 embryos per plate, on 560Y mediumfor 4 hours and then aligned within the 2.5 cm target zone inpreparation for bombardment. A plasmid vector DNA comprising thenucleotide sequence encoding the insecticidal protein operably linked toa promoter is precipitated onto 1.1 μm (average diameter) tungstenpellets using a CaCl₂ precipitation procedure as follows: 100 μlprepared tungsten particles in water; 10 μl (1 μg) DNA in Tris EDTAbuffer (1 μg total DNA); 100 μl 2.5 M CaCl₂ and 10 μl 0.1 M spermidine.

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 ml 100% ethanol and centrifugedfor 30 seconds. Again the liquid is removed, and 105 μl 100% ethanol isadded to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment. The sample plates are bombarded at level #4in a particle gun. All samples receive a single shot at 650 PSI, with atotal of ten aliquots taken from each tube of prepared particles/DNAFollowing bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for expression of a PtIP-83 polypeptideby assays known in the art, such as, for example, immunoassays andWestern blotting.

Transgenic maize plants positive for expression of the insecticidalproteins are tested for pesticidal activity using standard bioassaysknown in the art. Such methods include, for example, root excisionbioassays and whole plant bioassays. See, e.g., US Patent ApplicationPublication Number US 2003/0120054 and International Publication NumberWO 2003/018810.

Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/I Eriksson's Vitamin Mix (1000.times.SIGMA-1511), 0.5mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D and 2.88 g/lL-proline (brought to volume with D-I H₂O following adjustment to pH 5.8with KOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H₂O)and 8.5 mg/l silver nitrate (added after sterilizing the medium andcooling to room temperature). Selection medium (560R) comprises 4.0 g/lN6 basal salts (SIGMA C-1416), 1.0 ml/I Eriksson's Vitamin Mix(1000.times.SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose and 2.0mg/l 2,4-D (brought to volume with D-I H₂O following adjustment to pH5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with D-IH₂O) and 0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both addedafter sterilizing the medium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycinebrought to volume with polished D-I H₂O) (Murashige and Skoog, (1962)Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/lsucrose and 1.0 ml/I of 0.1 mM abscisic acid (brought to volume withpolished D-I H₂O after adjusting to pH 5.6); 3.0 g/l Gelrite (addedafter bringing to volume with D-I H₂O) and 1.0 mg/l indoleacetic acidand 3.0 mg/l bialaphos (added after sterilizing the medium and coolingto 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinicacid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL and 0.40 g/lglycine brought to volume with polished D-I H₂O), 0.1 g/l myo-inositoland 40.0 g/l sucrose (brought to volume with polished D-I H₂O afteradjusting pH to 5.6) and 6 g/l bacto-agar (added after bringing tovolume with polished D-I H₂O), sterilized and cooled to 60° C.

Example 9. Insect Control Efficacy of Stable Transformed Soybean andCorn Plants Against Broad Spectrum of Lepidopteran Insects

Leaf discs are excised from the transformed plants and tested forinsecticidal activity of PtIP-83 polypeptides against the Soy BeanLooper (SBL) (Chrysodeixis includens), Corn Earworm, (CEW) (Helicoverpazea), European Corn Borer (ECB) (Ostrinia nubilalis), Velvet BeanCaterpillar (VBC) (Anticarsia gemmatalis) and Fall Armyworm (Spodopterafrugiperda).

Example 10. Chimeras Between PtIP-83Aa (SEQ ID NO: 1) and PtIP-50Aa (SEQID NO: 34)

To generate single component active variants with diversified sequences,chimeras between PtIP-83Aa (SEQ ID NO: 1) and PtIP50Aa (SEQ ID NO: 34)were generated by multi-PCR fragments overlap assembly (Gibson AssemblyCloning Kit, New England Biolabs Inc.). A total of 6 chimeras wereconstructed: Table 13 shows the crossover points and the % sequenceidentity to PtIP-83Aa (SEQ ID NO: 1).

TABLE 13 Ending % Seq. Soybean Chimera poly- Beginning cross- iden. tolooper Designation nucleotide crossover over PtIP-83Aa active Chimera 1SEQ ID Q334 S473 94 No SEQ ID NO: 640 NO: 634 Chimera 2 SEQ ID G469 N61595 No SEQ ID NO: 641 NO: 635 Chimera 3 SEQ ID P610 E713 95 Yes SEQ IDNO: 642 NO: 636 Chimera 4 SEQ ID P754 E873 94 No SEQ ID NO: 643 NO: 637Chimera 5 SEQ ID F823 E873 98 Yes SEQ ID NO: 644 NO: 638 Chimera 6 SEQID Q334 E713 85 No SEQ ID NO: 645 NO: 639 Crossover position numbers arebased on the alignment shown in FIG. 3

The chimera genes were cloned into a plant transient expression vectorand SBL activity assays were performed as described in Example 4.

Example 11—Identification of Motifs Affecting the Protein Function ofPtIP-83Aa

To identify sequence space affecting protein structural stability andinsecticidal function of PtIP-83Aa, five unique motifs were identifiedby amino acid alignment among PtIP-83Aa, PtIP-50Aa (SEQ ID NO: 34),PtIP-50Ba (SEQ ID NO: 35), PtIP-50Bb (SEQ ID NO: 36), and PtIP-83Fa (SEQID NO: 3) (FIG. 4a-4d ). Three conserved motifs: amino acids V53-P66 ofSEQ ID NO: 1 were defined as Motif A, amino acids Q363-N373 of SEQ IDNO: 1 as Motif B and amino acids W556-A564 of SEQ ID NO: 1 as Motif C.Another two variable motifs were also picked: amino acids L646-W655 ofSEQ ID NO: 1 as Motif D and amino acids R771-Y786 of SEQ ID NO: 1 asMotif E. Saturation mutagenesis primers were designed for these fivemotifs as shown in Table 14, Table 15, Table 16, Table 17 and Table 18.Saturation mutagenesis was performed using Agilent's QuikChange®Lightning Site-Directed Mutagenesis Kit. Mutations were introduced byamplifying a plant expression vector containing the polynucleotide ofSEQ ID NO: 2 encoding the PtIP-83Aa polypeptide (SEQ ID NO: 1) usingcomplementing forward and reverse primers containing a NNK degeneratecodon at the targeted position. Amino acid substitutions at eachtargeted position were identified by DNA sequencing. Plant transientexpression and SBL activity assays were performed as described inexample 4. The amino acid substitutions identified in Motif A, Motif B,Motif C, Motif D, and Motif E of PtIP-83Aa (SEQ ID NO: 1) and the aminoacid substitutions with insecticidal activity are shown in Table 14,Table 15, Table 16, Table 17 and Table 18 respectively.

TABLE 14 Position Oligo name Primer Identified substitutions Activesubstitutions† V53 083SM-V53-F SEQ ID NO: 38 A, C, D, E, F, G, H, K, A,C, T L, N, P, Q, R, S, T, Y K54 083SM-K54-F SEQ ID NO: 39 A, C, D, E, F,G, H, I, A, C, D, E, G, H, I, L, 083SM-K54-R SEQ ID NO: 40 L, M, N, P,Q, R, S, T, M, N, Q, R, S, T V, W, Y R55 083SM-R55-F SEQ ID NO: 41 A, D,E, F, G, H, K, L, A, D, E, F, G, H, K, L, 083SM-R55-R SEQ ID NO: 42 M,N, P, Q, S, T, V, M, N, Q, S, T, V, W, Y W, Y L56 083SM-L56-F SEQ ID NO:43 A, D, E, F, G, I, M, N, E, F, I, M, T, V 083SM-L56-R SEQ ID NO: 44 P,Q, R, S, T, V, W, Y Y57 083SM-Y57-F SEQ ID NO: 45 A, C, D, E, G, H, I,K, C, I, L, M, T, V 083SM-Y57-R SEQ ID NO: 46 L, M, N, P, Q, R, S, T, VV58 083SM-V58-F SEQ ID NO: 47 A, C, D, F, G, H, I, C, I, L 083SM-V58-RSEQ ID NO: 48 K, L, N, P, Q, R, S, T, W, Y F59 083SM-F59-F SEQ ID NO: 49A, C, D, E, G, H, I, K, L, M, V, Y 083SM-F59-R SEQ ID NO: 50 L, M, N, P,Q, R, S, T, V, Y A60 083SM-A60-F SEQ ID NO: 51 C, D, E, F, G, H, I, L,C, G, S, T, V 083SM-A60-R SEQ ID NO: 52 M, N, P, Q, R, S, T, V, Y D61083SM-D61-F SEQ ID NO: 53 C, E, F, G, H, I, K, L, E, H, S 083SM-D61-RSEQ ID NO: 54 N, P, Q, R, S, T, V, W, Y V62 083SM-V62-F SEQ ID NO: 55 A,C, D, E, F, G, H, I, A, C, I, L, T 083SM-V62-R SEQ ID NO: 56 K, L, M, N,P, R, S, T, Y V63 083SM-V63-F SEQ ID NO: 57 A, C, G, H, I, K, L, M, A,C, I, L, M, T 083SM-V63-R SEQ ID NO: 58 N, P, Q, R, S, T, W, Y E64083SM-E64-F SEQ ID NO: 59 A, C, F, G, H, I, L, A, C, F, G, H, I, L, M,083SM-E64-R SEQ ID NO: 60 M, N, P, Q, R, S, T, N, Q, R, S, T, V, W, Y V,W, Y L65 083SM-L65-F SEQ ID NO: 61 A, C, D, F, G, H, I, A, C, F, H, I,M, N, Q, 083SM-L65-R SEQ ID NO: 62 M, N, P, Q, R, S, T, T, V, W V, W, YP66 083SM-P66-F SEQ ID NO: 63 A, C, D, E, F, G, I, K, D, G, M, Q, R083SM-P66-R SEQ ID NO: 64 L, M, N, Q, R, S, T, V, W, Y †Activesubstitutions: average activity score <= 60% of leaf disk eaten

TABLE 15 Position Oligo name Primer sequence Identified substitutionsActive substitutions Q363 083SM-Q363-F SEQ ID NO: 65 A, C, E, F, G, H,K, L, N, P, R, S, T, V, A, C, E, F, G, H, K, L, N, R, S, T, 083SM-Q363-RSEQ ID NO: 66 W V, W I364 083SM-I364-F SEQ ID NO: 67 A, C, D, E, F, G,H, K, L, M, N, P, Q, R, A, C, E, F, H, K, L, M, N, Q, S, T, 083SM-I364-RSEQ ID NO: 68 S, T, V, W, Y V, W, Y L365 083SM-L365-F SEQ ID NO: 69 A,E, F, G, H, I, K, M, N, P, R, S, T, V, A, E, F, G, H, I, K, M, N, R, V,W, 083SM-L365-R SEQ ID NO: 70 W, Y Y G366 083SM-G366-F SEQ ID NO: 71 A,C, E, F, H, I, K, L, M, N, P, R, S, T, A, C, F, H, I, K, L, M, N, S, T,V 083SM-G366-R SEQ ID NO: 72 V, W S367 083SM-S367-F SEQ ID NO: 73 A, C,D, E, F, G, H, I, L, M, N, P, Q, R, A, C, D, E, F, G, H, I, L, M, N, P,083SM-S367-R SEQ ID NO: 74 T, V, W Q, R, T, V, W Y368 083SM-Y368-F SEQID NO: 75 A, C, D, E, F, G, H, I, K, L, M, N, P, Q, A, C, D, E, F, G, H,I, K, L, M, N, 083SM-Y368-R SEQ ID NO: 76 R, S, T, V, W P, Q, R, S, T,V, W L369 083SM-L369-F SEQ ID NO: 77 A, C, D, F, G, H, I, M, N, P, R, S,T, V A, C, D, F, G, I, M, T, V 083SM-L369-R SEQ ID NO: 78 L370083SM-L370-F SEQ ID NO: 79 A, C, D, E, F, G, H, I, K, M, N, P, Q, R, A,C, D, E, F, G, H, I, K, M, Q, R, 083SM-L370-R SEQ ID NO: 80 S, T, V, W,Y S, T, V, W, Y Q371 083SM-Q371-F SEQ ID NO: 81 A, C, D, E, F, G, I, K,L, N, R, S, T, V, A, C, D, E, F, G, I, K, L, N, R, S, 083SM-Q371-R SEQID NO: 82 W T, V, W Q372 083SM-Q372-F SEQ ID NO: 83 A, C, D, F, G, H, I,L, N, R, S, T, V, Y A, C, D, F, G, H, I, L, N, R, S, V, 083SM-Q372-R SEQID NO: 84 Y N373 083SM-N373-F SEQ ID NO: 85 A, C, D, F, G, H, I, K, L,M, P, Q, R, S, A, C, D, F, G, H, I, K, Q, S, T, V, 083SM-N373-R SEQ IDNO: 86 T, V, W, Y W

TABLE 16 Position Oligo name Primer Sequence Identified substitutionsActive substitutions W556 083SM-W556-F SEQ ID NO: 87 A, C, D, F, G, I,K, L, M, N, P, Q, R, S, F, T, Y 083SM-W556-R SEQ ID NO: 88 T, V, Y R557083SM-R557-F SEQ ID NO: 89 C, D, G, H, I, K, L, M, N, P, Q, S, T, V, C,D, G, H, I, K, L, M, N, P, Q, S, 083SM-R557-R SEQ ID NO: 90 W, Y T, V,W, Y A558 083SM-A558-F SEQ ID NO: 91 C, D, F, G, H, I, K, L, N, P, Q, R,S, V, C, D, F, G, H, I, K, L, N, P, Q, R, 083SM-A558-R SEQ ID NO: 92 W,Y S, V, W, Y K559 083SM-K559-F SEQ ID NO: 93 A, C, F, G, H, I, L, N, P,Q, R, S, T, V, A, C, F, G, H, I, L, N, Q, R, S, T, 083SM-K559-R SEQ IDNO: 94 Y V, Y C560 083SM-C560-F SEQ ID NO: 95 A, D, F, G, H, I, K, L, M,N, P, Q, R, S, A, F, G, I, M, N, R, S, T, V, 083SM-C560-R SEQ ID NO: 96T, V, Y K561 083SM-K561-F SEQ ID NO: 97 A, C, D, E, F, G, H, I, L, M, N,P, R, S, A, C, D, E, F, G, H, I, L, M, N, R, 083SM-K561-R SEQ ID NO: 98T, V, Y S, T, V, Y N562 083SM-N562-F SEQ ID NO: 99 A, C, D, E, F, G, H,K, L, M, P, R, S, T, C, D, E, G, H, L, M, R, S, T, V, Y 083SM-N562-R SEQID NO: 100 V, W, Y V563 083SM-V563-F SEQ ID NO: 101 A, C, D, F, G, H, I,K, L, M, N, P, Q, R, A, C, D, F, H, I, L, M, N, Q, T, W 083SM-V563-R SEQID NO: 102 S, T, W A564 083SM-A564-F SEQ ID NO: 103 C, D, F, G, H, I, K,L, M, N, P, Q, R, S, C, G, M, Q, S, T, V, W, Y 083SM-A564-R SEQ ID NO:104 T, V, W, Y

TABLE 17 Position Oligo name Oligo Sequence Identified substitutionsActive substitutions L646 083SM-L646-F SEQ ID NO: 105 A, C, D, E, F, G,H, I, K, M, N, P, Q, R, A, C, G, I, M, N, Q, S, T, V 083SM-L646-R SEQ IDNO: 106 S, T, V, W, Y L647 083SM-L647-F SEQ ID NO: 107 A, D, F, G, H, I,K, M, N, P, Q, R, S, T, D, G, M, N, Q, T 083SM-L647-R SEQ ID NO: 108 V,W, Y M648 083SM-M648-F SEQ ID NO: 109 A, C, D, E, F, G, H, K, L, N, P,Q, R, S, A, C, D, E, F, G, H, K, L, N, P, Q, 083SM-M648-R SEQ ID NO: 110T, V, W, Y R, S, T, V, W, Y P649 083SM-P649-F SEQ ID NO: 111 A, C, D, E,F, G, H, I, K, L, M, N, Q, R, A, C, D, E, F, G, H, K, M, N, Q, R,083SM-P649-R SEQ ID NO: 112 S, T, V, W, Y S, T, W, Y T650 083SM-T650-FSEQ ID NO: 113 A, C, D, F, G, H, I, K, L, M, P, Q, R, S, A, C, D, F, G,H, I, K, L, M, P, Q, 083SM-T650-R SEQ ID NO: 114 V, Y R, S, V, Y E651083SM-E651-F SEQ ID NO: 115 A, C, D, G, H, I, K, L, M, N, P, Q, R, S, A,C, D, G, H, I, L, M, N, P, Q, R, 083SM-E651-R SEQ ID NO: 116 T, V, W, YS, T, V, Y L652 083SM-L652-F SEQ ID NO: 117 A, C, D, E, F, G, H, I, K,M, N, P, Q, R, C, F, I, K, M, P, R, S, T, V 083SM-L652-R SEQ ID NO: 118S, T, V, Y T653 083SM-T653-F SEQ ID NO: 119 C, D, E, F, G, H, I, K, L,P, R, S, V, W C, D, E, F, G, H, I, K, L, P, R, S, 083SM-T653-R SEQ IDNO: 120 V, W T654 083SM-T654-F SEQ ID NO: 121 A, C, F, H, I, K, L, M, N,P, Q, R, S, V, A, C, F, I, K, L, M, P, R, S, V, W, 083SM-T654-R SEQ IDNO: 122 W, Y Y W655 083SM-W655-F SEQ ID NO: 123 A, C, E, F, G, L, N, Q,R, S, T, V, Y F, Y 083SM-W655-R SEQ ID NO: 124

TABLE 18 Position Oligo name Primer Sequence Identified substitutionsActive substitutions R771 083SM-R771-F SEQ ID NO: 125 A, C, D, E, F, G,H, I, K, L, N, P, S, T, A, D, E, F, G, H, I, K, L, N, S, T, 083SM-R771-RSEQ ID NO: 126 V, W, Y V, W, Y R772 083SM-R772-F SEQ ID NO: 127 A, C, D,E, F, G, H, I, K, L, M, P, Q, S, A, C, D, E, F, G, H, I, K, L, M, P,083SM-R772-R SEQ ID NO: 128 T, V, W, Y Q, S, T, V, W, Y D773083SM-D773-F SEQ ID NO: 129 A, C, E, F, G, H, I, K, L, M, N, P, Q, R, A,E, F, G, H, I, K, L, M, N, Q, R, 083SM-D773-R SEQ ID NO: 130 S, T, V, W,Y S, T, V, W, Y Q774 083SM-Q774-F SEQ ID NO: 131 A, D, G, H, I, K, L, M,N, P, R, S, T, V, A, D, G, H, I, K, L, M, N, P, R, S, 083SM-Q774-R SEQID NO: 132 W, Y T, V, W, Y V775 083SM-V775-F SEQ ID NO: 133 A, C, D, E,G, H, I, L, M, N, P, Q, R, S, A, C, D, E, G, H, I, N, P, Q, R, S,083SM-V775-R SEQ ID NO: 134 T, Y T, Y L776 083SM-L776-F SEQ ID NO: 135A, C, D, E, F, G, H, I, K, N, P, Q, R, S, A, C, D, E, F, G, H, I, K, N,P, Q, 083SM-L776-R SEQ ID NO: 136 T, V, W, Y R, S, T, V, Y P777083SM-P777-F SEQ ID NO: 137 A, C, D, E, F, G, H, K, L, M, N, Q, R, S, A,C, D, E, F, G, H, K, L, M, N, Q, 083SM-P777-R SEQ ID NO: 138 T, V, W, YS, T, V, W, Y F778 083SM-F778-F SEQ ID NO: 139 A, D, G, H, I, K, L, M,N, P, Q, R, S, T, A, H, I, L, M, N, Q, S, V, W, Y 083SM-F778-R SEQ IDNO: 140 V, W, Y Q779 083SM-Q779-F SEQ ID NO: 141 A, C, D, E, F, G, H, I,K, L, N, P, R, S, A, C, D, E, G, H, K, L, N, P, R, S, 083SM-Q779-R SEQID NO: 142 T, V, W T, V A780 083SM-A780-F SEQ ID NO: 143 C, D, E, F, G,H, K, L, N, P, Q, R, S, T, C, N, P, Q, S 083SM-A780-R SEQ ID NO: 144 V,W, Y A781 083SM-A781-F SEQ ID NO: 145 C, D, E, F, G, H, I, L, M, N, P,Q, R, S, C, D, E, F, G, H, I, N, Q, R, S, T, 083SM-A781-R SEQ ID NO: 146T, V, W, Y V, W, Y A782 083SM-A782-F SEQ ID NO: 147 C, D, E, F, G, H, I,K, L, M, N, P, Q, R, C, D, E, F, G, H, I, K, M, P, Q, R, 083SM-A782-RSEQ ID NO: 148 S, T, V, W, Y S, T, V, W, Y P783 083SM-P783-F SEQ ID NO:149 A, C, D, E, F, G, H, I, L, M, N, Q, R, S, A, C, D, E, G, H, N, Q, R,S, T, V 083SM-P783-R SEQ ID NO: 150 T, V, Y L784 083SM-L784-F SEQ ID NO:151 A, C, D, E, F, G, H, I, K, M, N, P, Q, R, A, E, F, H, I, K, M, N, P,Q, S, T, 083SM-L784-R SEQ ID NO: 152 S, T, V, W, Y V, W N785083SM-N785-F SEQ ID NO: 153 A, C, E, F, G, H, I, K, L, M, P, Q, R, S, A,C, E, F, G, H, I, K, L, M, Q, R, 083SM-N785-R SEQ ID NO: 154 T, V, W, YS, T, V, W, Y Y786 083SM-Y786-F SEQ ID NO: 155 C, D, E, F, G, H, I, K,L, M, N, P, Q, R, F, I, L, W 083SM-Y786-R SEQ ID NO: 156 S, T, V, W

Example 12—PtIP-83Aa Variants with Multiple Amino Acid Substitutions inMotif A or Motif C

PtIP-83Aa variants with multiple amino acid substitutions within a motifwere constructed for Motif A and Motif C using either the QuikChange®Lightning Site-Directed Mutagenesis Kit (Agilent) (as described inExample 11), or by multi-PCR fragment overlap assembly (Gibson AssemblyCloning Kit, New England Biolabs Inc.) into a plant transient vectorcontaining the viral DMMV promoter. Primers used in constructingcombinations of mutations within an individual motif are summarized inTable 19. The resulting combinatory libraries were transformed intoAgrobacterium for plant transient expression and subsequent SBL activityassays as described in Example 4. Active variants, defined as having anaverage activity score of <=60% of leaf disk eaten, were sent forsequence identification.

TABLE 19 Library Primer Sequence Motif A 83M1-CombiF1CTCGAGGGAGCCGAGAAAGTGADGCRSYTCTATRTCTTKDSCGACRTCVTCGWG Combi -1VTCCCAGTCGTGGAATGGCGGTGG SEQ ID NO: 157 83M1-CombiF2CTCGAGGGAGCCGAGAAAGTGADGCRSYTCVTCRTCTTKDSCGACRTCVTCGWGVTCCCAGTCGTGGAATGGCGGTGG SEQ ID NO: 158 83M1-CombiRC1CCACCGCCATTCCACGACTGGGABCWCGABGAYGTCGSHMAAGAYATAGARSYGCHTCACTTTCTCGGCTCCCTCGAG SEQ ID NO: 159 83M1-CombiRC2CCACCGCCATTCCACGACTGGGABCWCGABGAYGTCGSHMAAGAYGABGARSYGCHTCACTTTCTCGGCTCCCTCGAG SEQ ID NO: 160 Motif A 83M1Cmb2-RCGGCCACCGCCATTCCACGACTGGTAGCWCGABAAYGTCGSHAAAAAYATACAA Combi -2SYGCHTCACTTTCTCGGCTCCCTCGAGCTG SEQ ID NO: 161 GZ550-83Aa-FAATCTCTCATCTAAGAGGCTGGATCCTAGGATGGCTCTCGTGGATTACGGC SEQ ID NO: 162GZ550-83Aa-R TGGCCAATCCAGAAGATGGACAAGTCTAGACTACTCTTCGTCGTGCCGCCAGSEQ ID NO: 163 83M1Cmb2-3p-F CTACCAGTCGTGGAATGGCGGTGGCCG SEQ ID NO: 164Motif C PtIP-83- GTCTCGGAGGTTCCGGTGTGGABSGBCMRGTGCAASWSCGTGGCTGCACTGGGTCombi MotifC-Combi- CGGGAGATG SEQ ID NO: 165 F1 PtIP-83-GTCTCGGAGGTTCCGGTGTGGCWSGBCMRGTGCAASWSCGTGGCTGCACTGGGT MotifC-Combi-CGGGAGATG SEQ ID NO: 166 F2 PtIP-83-GTCTCGGAGGTTCCGGTGTGGABSGBCMRGTGCAASMATGTGGCTGCACTGGGT MotifC-Combi-CGGGAGATG SEQ ID NO: 167 F3 PtIP-83-GTCTCGGAGGTTCCGGTGTGGCWSGBCMRGTGCAASMATGTGGCTGCACTGGGT MotifC-Combi-CGGGAGATG SEQ ID NO: 168 F4 GZ550-83Aa-FAATCTCTCATCTAAGAGGCTGGATCCTAGGATGGCTCTCGTGGATTACGGC SEQ ID NO: 162GZ550-83Aa-R TGGCCAATCCAGAAGATGGACAAGTCTAGACTACTCTTCGTCGTGCCGCCAGSEQ ID NO: 163 PtIP-83- CACCGGAACCTCCGAGACTTCCGTT SEQ ID NO: 171MotifC-Gibson- R

Two libraries with differing mutation rates were screened for Motif A.Motif A Combi-1 contains potential mutations at positions K54, R55, L56,Y57, V58, F59, A60, V62, V63, E64, and L65 in PtIP-83Aa (SEQ ID NO: 1):Motif A Combi-2 contains potential mutations at positions K54, R55, V58,A60, V62, V63, and E64 in PtIP-83Aa (SEQ ID NO: 1): From Motif ACombi-1, sequences were recovered for 5 active unique variants (96 totalscreened). From Motif A Combi-2, sequences were recovered for 59 activeunique variants (192 total screened). Table 20 summarizes the amino acidsubstitutions of the resulting active PtIP-83Aa Motif A variants ascompared to PtIP-83Aa.

TABLE 20 Motif A amino Additional Total # Variants DNA sequenceacid sequence mutations Mut. PtIP-83Aa SEQ ID NO: 2VKRLYVFADVVELP (a.a. 53- 0 SEQ ID NO: 1 66 of SEQ ID NO: 1)PtIP-83cmbM1-1 SEQ ID NO: 172 VK H LY I F C DV I ELP (a.a. 53- 4SEQ ID NO: 236 66 of SEQ ID NO: 236) PtIP-83cmbM1-2 SEQ ID NO: 173 VK HLY I F C DVVELP (a.a. 53- 3 SEQ ID NO: 237 66 of SEQ ID NO: 237)PtIP-83cmbM1-3 SEQ ID NO: 174 VK H LY I F S D II ELP (a.a. 53- 5SEQ ID NO: 238 66 of SEQ ID NO: 238) PtIP-83cmbM1-4 SEQ ID NO: 175 VK HLY I F T DV L ELP (a.a. 53- 4 SEQ ID NO: 239 66 of SEQ ID NO: 239)PtIP-83cmbM1-5 SEQ ID NO: 176 VK H LYVFAD IIV LP (a.a. 53- 4SEQ ID NO: 240 66 of SEQ ID NO: 240) PtIP-83cmbM1-6 SEQ ID NO: 177 VK HLYVFADV I ELP (a.a. 53- 2 SEQ ID NO: 241 66 of SEQ ID NO: 241)PtIP-83cmbM1-7 SEQ ID NO: 178 VK H LYVF G DV I ELP (a.a. 53- 3SEQ ID NO: 242 66 of SEQ ID NO: 242) PtIP-83cmbM1-8 SEQ ID NO: 179 VK HLYVF S DV I ELP (a.a. 53- 3 SEQ ID NO: 243 66 of SEQ ID NO: 243)PtIP-83cmbM1-9 SEQ ID NO: 180 VK H LYVF S DV LV LP (a.a. 53- 4SEQ ID NO: 244 66 of SEQ ID NO: 244) PtIP-83cmbM1-10 SEQ ID NO: 181 VK QL II F S D II ELP (a.a. 53- 6 SEQ ID NO: 245 66 of SEQ ID NO: 245)PtIP-83cmbM1-11 SEQ ID NO: 182 VK Q LY I F C D II ELP (a.a. 53- 5SEQ ID NO: 246 66 of SEQ ID NO: 246) PtIP-83cmbM1-12 SEQ ID NO: 183 VK QLY I FCDV L ELP (a.a. 53- 4 SEQ ID NO: 247 66 of SEQ ID NO: 247)PtIP-83cmbM1-13 SEQ ID NO: 184 VK Q LY I FGDV I ELP (a.a. 53- 4SEQ ID NO: 248 66 of SEQ ID NO: 248) PtIP-83cmbM1-14 SEQ ID NO: 185 VK QLY I F S D II ELP (a.a. 53- V24I 6 SEQ ID NO: 249 66 of SEQ ID NO: 249)PtIP-83cmbM1-15 SEQ ID NO: 186 VK Q LYVF C D IL ELP (a.a. 53- 4SEQ ID NO: 250 66 of SEQ ID NO: 250) PtIP-83cmbM1-16 SEQ ID NO: 187 VK QLYVF S D IL ELP (a.a. 53- 4 SEQ ID NO: 251 66 of SEQ ID NO: 251)PtIP-83cmbM1-17 SEQ ID NO: 188 VK Q LYVF S DV LV LP (a.a. 53- 4SEQ ID NO: 252 66 of SEQ ID NO: 252) PtIP-83cmbM1-18 SEQ ID NO: 189 VKRF Y I FAD I VELP (a.a. 53- 3 SEQ ID NO: 253 66 of SEQ ID NO: 253)PtIP-83cmbM1-19 SEQ ID NO: 190 VKR F Y I FSD II ELP (a.a. 53- 5SEQ ID NO: 254 66 of SEQ ID NO: 254) PtIP-83cmbM1-20 SEQ ID NO: 191 VKRF YVF S D I VELP (a.a. 53- 3 SEQ ID NO: 255 66 of SEQ ID NO: 255)PtIP-83cmbM1-21 SEQ ID NO: 192 VKRL VILG D IIVV P (a.a. 53- 8SEQ ID NO: 256 66 of SEQ ID NO: 256) PtIP-83cmbM1-22 SEQ ID NO: 193VKRLY I FAD II ELP (a.a. 53- 3 SEQ ID NO: 257 66 of SEQ ID NO: 257)PtIP-83cmbM1-23 SEQ ID NO: 194 VKRLY I FCD IIV LP (a.a. 53- 5SEQ ID NO: 258 66 of SEQ ID NO: 258) PtIP-83cmbM1-24 SEQ ID NO: 195VKRLY I F C D I VELP (a.a. 53- 3 SEQ ID NO: 259 66 of SEQ ID NO: 259)PtIP-83cmbM1-25 SEQ ID NO: 196 VKRLY I F G D II ELP (a.a. 53- P74A 5SEQ ID NO: 260 66 of SEQ ID NO: 260) PtIP-83cmbM1-26 SEQ ID NO: 197VKRLY I F G DV I ELP (a.a. 53- 3 SEQ ID NO: 261 66 of SEQ ID NO: 261)PtIP-83cmbM1-27 SEQ ID NO: 198 VKRLY I F S D IIV LP (a.a. 53- 5SEQ ID NO: 262 66 of SEQ ID NO: 262) PtIP-83cmbM1-28 SEQ ID NO: 199VKRLY I F S D IL ELP (a.a. 53- 4 SEQ ID NO: 263 66 of SEQ ID NO: 263)PtIP-83cmbM1-29 SEQ ID NO: 200 VKRLY I F S DV IV LP (a.a. 53- 4SEQ ID NO: 264 66 of SEQ ID NO: 264) PtIP-83cmbM1-30 SEQ ID NO: 201VKRLY I F T DV I ELP (a.a. 53- 3 SEQ ID NO: 265 66 of SEQ ID NO: 265)PtIP-83cmbM1-31 SEQ ID NO: 202 VKRLYVF C D II ELP (a.a. 53- 3SEQ ID NO: 266 66 of SEQ ID NO: 266) PtIP-83cmbM1-32 SEQ ID NO: 203VKRLYVF C D IIV LP (a.a. 53- 4 SEQ ID NO: 267 66 of SEQ ID NO: 267)PtIP-83cmbM1-33 SEQ ID NO: 204 VKRLYVF G D I VELP (a.a. 53- 2SEQ ID NO: 268 66 of SEQ ID NO: 268) PtIP-83cmbM1-34 SEQ ID NO: 205VKRLYVF G DVVELP (a.a. 53- 1 SEQ ID NO: 269 66 of SEQ ID NO: 269)PtIP-83cmbM1-35 SEQ ID NO: 206 VKRLYVF S D II ELP (a.a. 53- 3SEQ ID NO: 270 66 of SEQ ID NO: 270) PtIP-83cmbM1-36 SEQ ID NO: 207VKRLYVF S D I VELP (a.a. 53- 2 SEQ ID NO: 271 66 of SEQ ID NO: 271)PtIP-83cmbM1-37 SEQ ID NO: 208 VKRLYVF S DV I ELP (a.a. 53- 2SEQ ID NO: 272 66 of SEQ ID NO: 272) PtIP-83cmbM1-38 SEQ ID NO: 209VKRLYVF T DV IV LP (a.a. 53- 3 SEQ ID NO: 273 66 of SEQ ID NO: 273)PtIP-83cmbM1-39 SEQ ID NO: 210 VKRLYVF T DVV V LP (a.a. 53- 2SEQ ID NO: 274 66 of SEQ ID NO: 274) PtIP-83cmbM1-40 SEQ ID NO: 211 V MHLY I FADV I ELP (a.a. 53- 4 SEQ ID NO: 275 66 of SEQ ID NO: 275)PtIP-83cmbM1-41 SEQ ID NO: 212 V MQ LY I F C D IL ELP (a.a. 53- P74T 7SEQ ID NO: 276 66 of SEQ ID NO: 276) PtIP-83cmbM1-42 SEQ ID NO: 213 V MRLY I FADVV V LP (a.a. 53- 3 SEQ ID NO: 277 66 of SEQ ID NO: 277)PtIP-83cmbM1-43 SEQ ID NO: 214 V M RLY I F C DV I ELP (a.a. 53- 4SEQ ID NO: 278 66 of SEQ ID NO: 278) PtIP-83cmbM1-44 SEQ ID NO: 215 V MRLYVF C DI I ELP (a.a. 53- 4 SEQ ID NO: 279 66 of SEQ ID NO: 279)PtIP-83cmbM1-45 SEQ ID NO: 216 V M RLYVF C D ILV LP (a.a. 53- 5SEQ ID NO: 280 66 ofSEQID NO: 280) PtIP-83cmbM1-46 SEQ ID NO: 217 V MRLYVF S D IIV LP (a.a. 53- 5 SEQ ID NO: 281 66 ofSEQID NO: 281)PtIP-83cmbM1-47 SEQ ID NO: 218 V RH LYIFAD II ELP (a.a. 53- 5SEQ ID NO: 282 66 of SEQ ID NO: 282) PtIP-83cmbM1-48 SEQ ID NO: 219 V RHLY I FADVVELP (a.a. 53- 3 SEQ ID NO: 283 66 of SEQ ID NO: 283)PtIP-83cmbM1-49 SEQ ID NO: 220 V RH LY I F C DV I ELP (a.a. 53- 5SEQ ID NO: 284 66 of SEQ ID NO: 284) PtIP-83cmbM1-50 SEQ ID NO: 221 V RHLY I F S DV I ELP (a.a. 53- 5 SEQ ID NO: 285 66 of SEQ ID NO: 285)PtIP-83cmbM1-51 SEQ ID NO: 222 V RH LY I F S DVVELP (a.a. 53- 4SEQ ID NO: 286 66 of SEQ ID NO: 286) PtIP-83cmbM1-52 SEQ ID NO: 223 V RHLYVF T DV L ELP (a.a. 53- 4 SEQ ID NO: 287 66 of SEQ ID NO: 287)PtIP-83cmbM1-53 SEQ ID NO: 224 V RQ LY I F C DV IV LP (a.a. 53- 6SEQ ID NO: 288 66 of SEQ ID NO: 288) PtIP-83cmbM1-54 SEQ ID NO: 225 V RQLY I F S DVV V LP (a.a. 53- 5 SEQ ID NO: 289 66 of SEQ ID NO: 289)PtIP-83cmbM1-55 SEQ ID NO: 226 V RQ LYVF C DV LV LP (a.a. 53- 5SEQ ID NO: 290 66 of SEQ ID NO: 290) PtIP-83cmbM1-56 SEQ ID NO: 227 V RRLY I FAD IL ELP (a.a. 53- 4 SEQ ID NO: 291 66 of SEQ ID NO: 291)PtIP-83cmbM1-57 SEQ ID NO: 228 V R RLY I FAD IV ELP (a.a. 53- P74A 4SEQ ID NO: 292 66 of SEQ ID NO: 292) PtIP-83cmbM1-58 SEQ ID NO: 229 V RRLY I F G D IV ELP (a.a. 53- 4 SEQ ID NO: 293 66 of SEQ ID NO: 293)PtIP-83cmbM1-59 SEQ ID NO: 230 V R RLY I F T D II ELP (a.a. 53- 5SEQ ID NO: 294 66 of SEQ ID NO: 294) PtIP-83cmbM1-60 SEQ ID NO: 231 V RRLYVFAD IID LP (a.a. 53- 4 SEQ ID NO: 295 66 of SEQ ID NO: 295)PtIP-83cmbM1-61 SEQ ID NO: 232 V R RLYVFAD I V V LP (a.a. 53- 3SEQ ID NO: 296 66 of SEQ ID NO: 296) PtIP-83cmbM1-62 SEQ ID NO: 233 V RRLYVF C DVV V LP (a.a. 53- 3 SEQ ID NO: 297 66 of SEQ ID NO: 297)PtIP-83cmbM1-63 SEQ ID NO: 234 V R RLYVF T D II ELP (a.a. 53- 4SEQ ID NO: 298 66 of SEQ ID NO: 298) PtIP-83cmbM1-64 SEQ ID NO: 235 V RRLYVF T DV LV LP (a.a. 53- 4 SEQ ID NO: 299 66 of SEQ ID NO: 299)

A single combination library of Motif C containing potential mutationsat positions R557, A558, K559, K561, and N562 of PtIP-83Aa (SEQ IDNO: 1) was screened. From a total of 233 variants screened from Motif CCombi, 34 active unique sequence variants were identified. Table 21summarizes the amino acid substitutions of the resulting activePtIP-83Aa Motif C variants as compared to PtIP-83Aa.

TABLE 21 Additional Total Variants DNA sequence AA sequence mutationsMutations PtIP-83Aa SEQ ID NO: 2 WRAKCKNVA (a.a. 556- 0 SEQ ID NO: 1564 of SEQ ID NO: 1) PtIP-83cmbM3-1 SEQ ID NO: 300 W H A R CK SVA (a.a. 556- 3 SEQ ID NO: 334 564 of SEQ ID NO: 334) PtIP-83cmbM3-2SEQ ID NO: 301 W HG KCKNVA (a.a. 556- 2 SEQ ID NO: 335564 of SEQ ID NO: 335) PtIP-83cmbM3-3 SEQ ID NO: 302 W I AKCK CVA (a.a. 556- 2 SEQ ID NO: 336 564 of SEQ ID NO: 336) PtIP-83cmbM3-4SEQ ID NO: 303 W I AKCK S VA (a.a. 556- 2 SEQ ID NO: 337564 of SEQ ID NO: 337) PtIP-83cmbM3-5 SEQ ID NO: 304 W IGKCKNVA (a.a. 556- 2 SEQ ID NO: 338 564 of SEQ ID NO: 338) PtIP-83cmbM3-6SEQ ID NO: 305 W IG KCK S VA (a.a. 556- 3 SEQ ID NO: 339564 of SEQ ID NO: 339) PtIP-83cmbM3-7 SEQ ID NO: 306 W IG KCN NVA (a.a. 556- 3 SEQ ID NO: 340 564 of SEQ ID NO: 340) PtIP-83cmbM3-8SEQ ID NO: 307 W IV KCK S VA (a.a. 556- 3 SEQ ID NO: 341564 of SEQ ID NO: 341) PtIP-83cmbM3-9 SEQ ID NO: 308 W L A RCKNVA (a.a. 556- 2 SEQ ID NO: 342 564 of SEQ ID NO: 342) PtIP-83cmbM3-10SEQ ID NO: 309 W L A R CK S VA (a.a. 556- 3 SEQ ID NO: 343564 of SEQ ID NO: 343) PtIP-83cmbM3-11 SEQ ID NO: 310 W LGRCKNVA (a.a. 556- 3 SEQ ID NO: 344 564 of SEQ ID NO: 344) PtIP-83cmbM3-12SEQ ID NO: 311 W LGR CK S VA (a.a. 556- 4 SEQ ID NO: 345564 of SEQ ID NO: 345) PtIP-83cmbM3-13 SEQ ID NO: 312 W LV KCK CVA (a.a. 556- 3 SEQ ID NO: 346 564 of SEQ ID NO: 346) PtIP-83cmbM3-14SEQ ID NO: 313 W M AKCKNVA (a.a. 556- A502S 2 SEQ ID NO: 347564 of SEQ ID NO: 347) PtIP-83cmbM3-15 SEQ ID NO: 314 W MGR CK SVA (a.a. 556- 4 SEQ ID NO: 348 564 of SEQ ID NO: 348) PtIP-83cmbM3-16SEQ ID NO: 315 W MV KCKNVA (a.a. 556- 2 SEQ ID NO: 349564 of SEQ ID NO: 349) PtIP-83cmbM3-17 SEQ ID NO: 316 W MV KCK SVA (a.a. 556- 3 SEQ ID NO: 350 564 of SEQ ID NO: 350) PtIP-83cmbM3-18SEQ ID NO: 317 W MVR CKNVA (a.a. 556- 3 SEQ ID NO: 351564 of SEQ ID NO: 351) PtIP-83cmbM3-19 SEQ ID NO: 318 W Q A R CK HVA (a.a. 556- 3 SEQ ID NO: 352 564 of SEQ ID NO: 352) PtIP-83cmbM3-20SEQ ID NO: 319 W Q A R CKNVA (a.a. 556- K505N 3 SEQ ID NO: 353564 of SEQ ID NO: 353) PtIP-83cmbM3-21 SEQ ID NO: 320 W QGRCKNVA (a.a. 556- 3 SEQ ID NO: 354 564 of SEQ ID NO: 354) PtIP-83cmbM3-22SEQ ID NO: 321 W QVR CK S VA (a.a. 556- 4 SEQ ID NO: 355564 of SEQ ID NO: 355) PtIP-83cmbM3-23 SEQ ID NO: 322 WRA RCKNVA (a.a. 556- T573A 2 SEQ ID NO: 356 564 of SEQ ID NO: 356)PtIP-83cmbM3-24 SEQ ID NO: 323 WR G KCK S VA (a.a. 556- 2 SEQ ID NO: 357564 of SEQ ID NO: 357) PtIP-83cmbM3-25 SEQ ID NO: 324 WR GR CK TVA (a.a. 556- 3 SEQ ID NO: 358 564 of SEQ ID NO: 358) PtIP-83cmbM3-26SEQ ID NO: 325 W S A R CK S VA (a.a. 556- 3 SEQ ID NO: 359564 of SEQ ID NO: 359) PtIP-83cmbM3-27 SEQ ID NO: 326 W SV KCK HVA (a.a. 556- SEQ ID NO: 360 564 of SEQ ID NO: 360) PtIP-83cmbM3-28SEQ ID NO: 327 W TGR CK T VA (a.a. 556- SEQ ID NO: 361564 of SEQ ID NO: 361) PtIP-83cmbM3-29 SEQ ID NO: 328 W TGR CN HVA (a.a. 556- R568Q 6 SEQ ID NO: 362 564 of SEQ ID NO: 362)PtIP-83cmbM3-30 SEQ ID NO: 329 W TV KCKNVA (a.a. 556- 2 SEQ ID NO: 363564 of SEQ ID NO: 363) PtIP-83cmbM3-31 SEQ ID NO: 330 W TV KCK SVA (a.a. 556- 3 SEQ ID NO: 364 564 of SEQ ID NO: 364) PtIP-83cmbM3-32SEQ ID NO: 331 W TVR CKNVA (a.a. 556- 3 SEQ ID NO: 365564 of SEQ ID NO: 365) PtIP-83cmbM3-33 SEQ ID NO: 332WRARCKHVA (a.a. 556- 2 SEQ ID NO: 366 564 of SEQ ID NO: 366)PtIP-83cmbM3-34 SEQ ID NO: 333 WIGRCKSVA (a.a. 556- 4 SEQ ID NO: 367564 of SEQ ID NO: 367)

Example 13—PtIP-83Aa Variants with Multiple Amino Acid Substitutions inMotif A and Motif C

Additional sequence diversity was created by combining active Motif Acombinations with active Motif C combinations. Twenty four unique activeMotif A combination variants and 11 unique active Motif C combinationvariants were selected for construction of a Motif A×Motif C Library(Table 22).

TABLE 22 Sequence variants used for construction of Motif A x Motif CCombination Library Motif A Motif C PtIP-83cmbM1-1 PtIP-83cmbM1-32PtIP-83cmbM3-2 SEQ ID NO: 236 SEQ ID NO: 267 SEQ ID NO: 335PtIP-83cmbM1-2 PtIP-83cmbM1-35 PtIP-83cmbM3-5 SEQ ID NO: 237 SEQ ID NO:270 SEQ ID NO: 338 PtIP-83cmbM1-4 PtIP-83cmbM1-36 PtIP-83cmbM3-9 SEQ IDNO: 239 SEQ ID NO: 271 SEQ ID NO: 342 PtIP-83cmbM1-6 PtIP-83cmbM1-37PtIP-83cmbM3-11 SEQ ID NO: 241 SEQ ID NO: 272 SEQ ID NO: 344PtIP-83cmbM1-12 PtIP-83cmbM1-44 PtIP-83cmbM3-16 SEQ ID NO: 247 SEQ IDNO: 279 SEQ ID NO: 349 PtIP-83cmbM1-15 PtIP-83cmbM1-49 PtIP-83cmbM3-19SEQ ID NO: 250 SEQ ID NO: 284 SEQ ID NO: 352 PtIP-83cmbM1-16PtIP-83cmbM1-51 PtIP-83cmbM3-20 SEQ ID NO: 251 SEQ ID NO: 286 SEQ ID NO:353 PtIP-83cmbM1-18 PtIP-83cmbM1-55 PtIP-83cmbM3-23 SEQ ID NO: 253 SEQID NO: 290 SEQ ID NO: 356 PtIP-83cmbM1-21 PtIP-83cmbM1-56PtIP-83cmbM3-24 SEQ ID NO: 256 SEQ ID NO: 291 SEQ ID NO: 357PtIP-83cmbM1-22 PtIP-83cmbM1-60 PtIP-83cmbM3-25 SEQ ID NO: 257 SEQ IDNO: 295 SEQ ID NO: 358 PtIP-83cmbM1-30 PtIP-83cmbM1-62 PtIP-83cmbM3-30SEQ ID NO: 265 SEQ ID NO: 297 SEQ ID NO: 363 PtIP-83cmbM1-31PtIP-83cmbM1-64 SEQ ID NO: 266 SEQ ID NO: 299

Plasmid DNA was isolated from these variants and pooled by motif. MotifA combinations were PCR amplified from the Motif A pool using thefollowing primers: AATCTCTCATCTAAGAGGCTGGATCCTAGGATGGCTCTCGTGGATTACGGC(SEQ ID NO: 630) and GCAGCCACAACCTCCATCACAGC (SEQ ID NO: 631). Motif Ccombinations were PCR amplified from the Motif C pool using thefollowing primers: TGGCCAATCCAGAAGATGGACAAGTCTAGACTACTCTTCGTCGTGCCGCCAG(SEQ ID NO: 632) and GCTGTGATGGAGGTTGTGGCTGC (SEQ ID NO: 633). The twoPCR products were then assembled by multi-PCR fragments overlap (GibsonAssembly Cloning Kit, New England Biolabs Inc) into a plant transientvector containing the viral DMMV promoter. 94 variants from this librarywere screened by plant transient expression and SBL activity assay asdescribed in Example 4. 30 unique active variants, defined as having anaverage activity score of <=60% of leaf disk eaten, were identified bysequencing. Table 23 summarizes the amino acid substitutions of theresulting active PtIP-83Aa Motif A×Motif C variants as compared toPtIP-83Aa.

TABLE 23 Motif C Add. Total Variant DNA sequence Motif A sequencesequence Mut. Mut. PtIP-83Aa SEQ ID NO: 2 VKRLYVFADVVELP WRAKCKNVA 0SEQ ID NO: 1 (a.a. 53-66 of (a.a. 556- SEQ ID NO: 1) 564 of SEQ IDNO: 1) PtIP-83cmbM1xM3-1 SEQ ID NO: 368 VK H LY I F T DV L ELP W HGKCKNVA S533F 7 SEQ ID NO: 398 (a.a. 53-66 of (a.a. 556- SEQ ID NO: 398)564 of SEQ ID NO: 398) PtIP-83cmbM1xM3-2 SEQ ID NO: 369 VK H LY I F T DVL ELP W L A R CKNVA 6 SEQ ID NO: 399 (a.a. 53-66 of (a.a. 556-SEQ ID NO: 399) 564 of SEQ ID NO: 399) PtIP-83cmbM1xM3-3 SEQ ID NO: 370VK H LY I F T DV L ELP W Q A R CK H VA 7 SEQ ID NO: 400 (a.a. 53-66 of(a.a. 556- SEQ ID NO: 400) 564 of SEQ ID NO: 400) PtIP-83cmbM1xM3-4SEQ ID NO: 371 VK H LYVFADV I ELP W Q A R CK H VA 5 SEQ ID NO: 401(a.a. 53-66 of (a.a. 556- SEQ ID NO: 401) 564 of SEQ ID NO: 401)PtIP-83cmbM1xM3-5 SEQ ID NO: 372 VK H LYVFADV I ELP WRA R CKNVA 3SEQ ID NO: 402 (a.a. 53-66 of (a.a. 556- SEQ ID NO: 402) 564 of SEQ IDNO: 402) PtIP-83cmbM1xM3-6 SEQ ID NO: 373 VK Q LY I F C DV L ELP W Q A RCKNVA 6 SEQ ID NO: 403 (a.a. 53-66 of (a.a. 556- SEQ ID NO: 403)564 of SEQ ID NO: 403) PtIP-83cmbM1xM3-7 SEQ ID NO: 374 VKR F Y I FAD IVELP W IG KCKNVA 5 SEQ ID NO: 404 (a.a. 53-66 of (a.a. 556-SEQ ID NO: 404) 564 of SEQ ID NO: 404) PtIP-83cmbMxM3-8 SEQ ID NO: 375VKR F Y I FAD I VELP W TV KCKNVA 5 SEQ ID NO: 405 (a.a. 53 66 of(a.a. 556- SEQ ID NO: 405) 564 of SEQ ID NO: 405) PtIP-83cmbM1xM3-9SEQ ID NO: 376 VKRLY I FAD II ELP W MV KCKNVA 5 SEQ ID NO: 406(a.a. 53-66 of (a.a. 556- SEQ ID NO: 406) 564 of SEQ ID NO: 406)PtIP-83cmbM1xM3-10 SEQ ID NO: 377 VKRLY I FAD II ELP W Q A R CKNVA 5SEQ ID NO: 407 (a.a. 53-66 of (a.a. 556- SEQ ID NO: 407) 564 of SEQ IDNO: 407) PtIP-83cmbM1xM3-11 SEQ ID NO: 378 VKRLY I FAD II ELP WR G KCK SVA K52E, 7 SEQ ID NO: 408 (a.a. 53-66 of (a.a. 556- K505NSEQ ID NO: 408) 564 of SEQ ID NO: 408) PtIP-83cmbM1xM3-12 SEQ ID NO: 379VKRLY I FAD II ELP WR GR CK T VA 6 SEQ ID NO: 409 (a.a. 53-66 of(a.a. 556- SEQ ID NO: 409) 564 of SEQ ID NO: 409) PtIP-83cmbM1xM3-13SEQ ID NO: 380 VKRLY I F T DV I ELP W IG KCKNVA 5 SEQ ID NO: 410(a.a. 53-66 of (a.a. 556- SEQ ID NO: 410) 564 of SEQ ID NO: 410)PtIP-83cmbM1xM3-14 SEQ ID NO: 381 VKRLYVF C D II ELP W L A R CKNVA 5SEQ ID NO: 411 (a.a. 53-66 of (a.a. 556- SEQ ID NO: 411) 564 of SEQ IDNO: 411) PtIP-83cmbM1xM3-15 SEQ ID NO: 382 VKRLYVF S D II ELP W IGKCKNVA 5 SEQ ID NO: 412 (a.a. 53-66 of (a.a. 556- SEQ ID NO: 412)564 of SEQ ID NO: 412) PtIP-83cmbM1xM3-16 SEQ ID NO: 383 VKRLYVF S D IIELP WR GR CK T VA 6 SEQ ID NO: 413 (a.a. 53-66 of (a.a. 556-SEQ ID NO: 413) 564 of SEQ ID NO: 413) PtIP-83cmbM1xM3-17 SEQ ID NO: 384VKRLYVF S DV I ELP WR G KCK S VA 4 SEQ ID NO: 414 (a.a. 53-66 of(a.a. 556- SEQ ID NO: 414) 564 of SEQ ID NO: 414) PtIP-83cmbM1xM3-18SEQ ID NO: 385 VKRLYVF S DV I ELP WR V KCKNVA 3 SEQ ID NO: 415(a.a. 53-66 of (a.a. 556- SEQ ID NO: 415) 564 of SEQ ID NO: 415)PtIP-83cmbM1xM3-19 SEQ ID NO: 386 V M RLY I FADVVELP WR G KCK S VA 4SEQ ID NO: 416 (a.a. 53-66 of (a.a. 556- SEQ ID NO: 416) 564 of SEQ IDNO: 416) PtIP-83cmbM1xM3-20 SEQ ID NO: 387 V RH LY I F C DV I ELP W IGKCKNVA 7 SEQ ID NO: 417 (a.a. 53-66 of (a.a. 556- SEQ ID NO: 417)564 of SEQ ID NO: 417) PtIP-83cmbM1xM3-21 SEQ ID NO: 388 V RH LY I F SDVVELP W MV KCKNVA 6 SEQ ID NO: 418 (a.a. 53-66 of (a.a. 556-SEQ ID NO: 418) 564 of SEQ ID NO: 418) PtIP-83cmbM1xM3-22 SEQ ID NO: 389V RH LY I F S DVVELP WR G KCK S VA K505N 7 SEQ ID NO: 419 (a.a. 53-66 of(a.a. 556- SEQ ID NO: 419) 564 of SEQ ID NO: 419) PtIP-83cmbM1xM3-23SEQ ID NO: 390 V RQ LYVF C DV LV LP W HG KCKNVA 7 SEQ ID NO: 420(a.a. 53-66 of (a.a. 556- SEQ ID NO: 420) 564 of SEQ ID NO: 420)PtIP-83cmbM1xM3-24 SEQ ID NO: 391 V RQ LYVF C DV LV LP W LGR CKNVA 8SEQ ID NO: 421 (a.a. 53-66 of (a.a. 556- SEQ ID NO: 421) 564 of SEQ IDNO: 421) PtIP-83cmbM1xM3-25 SEQ ID NO: 392 V RQ LYVF C DV LV LP WR G KCKS VA M1I 8 SEQ ID NO: 422 (a.a. 53-66 of (a.a. 556- SEQ ID NO: 422)564 of SEQ ID NO: 422) PtIP-83cmbM1xM3-26 SEQ ID NO: 393 V R RLY I FADIL ELP W MV KCKNVA 6 SEQ ID NO: 423 (a.a. 53-66 of (a.a. 556-SEQ ID NO: 423) 564 of SEQ ID NO: 423) PtIP-83cmbM1xM3-27 SEQ ID NO: 394V R RLYVF C DVV V LP WRA R CKNVA 4 SEQ ID NO: 424 (a.a. 53-66 of(a.a. 556- SEQ ID NO: 424) 564 of SEQ ID NO: 424) PtIP-83cmbM1xM3-28SEQ ID NO: 395 V R RLYVF T DV LV LP W IG KCKNVA 6 SEQ ID NO: 425(a.a. 53-66 of (a.a. 556- SEQ ID NO: 425) 564 of SEQ ID NO: 425)PtIP-83cmbM1xM3-29 SEQ ID NO: 396 V R RLYVF T DV LV LP WRA R CKNVA 5SEQ ID NO: 426 (a.a. 53-66 of (a.a. 556- SEQ ID NO: 426) 564 of SEQ IDNO: 426) PtIP-83cmbM1xM3-30 SEQ ID NO: 397 V R RLYVF T DV LV LP W TVKCKNVA 6 SEQ ID NO: 427 (a.a. 53-66 of (a.a. 556- SEQ ID NO: 427)564 of SEQ ID NO: 427)

Example 14 PtIP-83Aa Variants with Multiple Amino Acid Substitutions

To create variants of PtIP-83Aa (SEQ ID NO: 1) with multiple amino acidchanges, variant libraries were generated by family shuffling (Chia-ChunJ. Chang et al, 1999, Nature Biotechnology 17, 793-797) of thepolynucleotide (SEQ ID NO: 2) encoding PtIP-83Aa (SEQ ID NO: 1) and thepolynucleotide (SEQ ID NO: 2) encoding PtIP-83Cb (SEQ ID NO: 7).Mutation rates of the libraries were controlled by varying the regionsof PtIP-83Aa (SEQ ID NO: 1) and PtIP-83Cb (SEQ ID NO: 7) which wereincluded in the shuffling library reaction. Five libraries weregenerated between the two variants. In the first library (83FS-1), thefull length gene sequence from both PtIP-83Aa (SEQ ID NO: 2) andPtIP-83Cb (SEQ ID NO: 8) were included in the shuffling reaction. In thesecond library (83FS-2), fragments containing nt: 1114-2604 of SEQ IDNO: 2 (encoding PtIP-83Aa (SEQ ID NO: 1)) and nt: 1072-2559 of SEQ IDNO: 8 (encoding PtIP-83Cb (SEQ ID NO: 7)) were shuffled. The shuffledfragments were then combined with nt: 1-1113 of SEQ ID NO: 2 (encodingPtIP-83Aa (SEQ ID NO: 1)) by Gibson Assembly (New England Biolabs). In athird library (83FS-3), the 5′ and 3′ regions of the two variants wereshuffled in separate reactions. The 5′ reaction, containing shuffledfragments from nt: 1-1113 of SEQ ID NO: 2 (encoding PtIP-83Aa (SEQ IDNO: 1)) and nt:1-1071 of SEQ ID NO: 8 (encoding PtIP-83Cb (SEQ ID NO:7)), and the 3′ reaction, containing shuffled fragments from nt:1114-2604 of SEQ ID NO: 2 (encoding PtIP-83Aa (SEQ ID NO: 1)) and nt:1072-2559 of SEQ ID NO: 8 (encoding PtIP-83Cb (SEQ ID NO: 7)), were thencombined by Gibson Assembly. In a fourth library (83FS-4), fragmentsfrom the 5′ region of each variant (nt: 1-1113 of SEQ ID NO: 2 (encodingPtIP-83Aa (SEQ ID NO: 1)) and nt:1-1071 of SEQ ID NO: 8 (encodingPtIP-83Cb (SEQ ID NO: 7)) were shuffled with additional primers spikedinto the assembly reaction to promote crossovers between the twovariants. The shuffled 5′ region was then combined by Gibson assemblywith the 3′ region of either SEQ ID NO: 2 (encoding PtIP-83Aa (SEQ IDNO: 1)) or SEQ ID NO: 8 (encoding PtIP-83Cb (SEQ ID NO: 7)). In thefifth library (83FS-5), single crossover variants were generatedmanually between SEQ ID NO: 2 (encoding PtIP-83Aa (SEQ ID NO: 1)) andSEQ ID NO: 8 (encoding PtIP-83Cb (SEQ ID NO: 7)) using Gibson assembly.

All shuffled libraries were cloned into a plant transient expressionvector containing the viral dMMV promoter for subsequent transientexpression in bush bean and SBL activity assays as described in Example4. Active variants, defined as having an average activity score of ≤60%of leaf disk eaten, were sent for sequence identification. In library83FS-1, 197 variants were screened and 45 active unique variants weresequence identified. In library 83FS-2, 96 variants were screened and 32active unique variants were sequence identified. In library 83FS-3, 192variants were screened and 13 active unique variants were sequenceidentified. In library 83FS-4, 96 variants were screened and 6 uniqueactive variants were sequence identified. In library 83FS-5, four of theeleven constructed single cross variants were found to be active.

Sequence identity of active variants to PtIP-83Aa was calculated usingthe Needleman-Wunsch algorithm, as implemented in the Needle program(EMBOSS tool suite). The percent identity compared to PtIP-83Aa (SEQ IDNO: 1), variant designation, nucleotide sequences, and amino acidsequences of the resulting active PtIP-83Aa polypeptide variants fromall libraries are summarized in Table 24. Table 25 summarizes the %identity of the active variants compared to PtIP-83Aa (SEQ ID NO: 1),the number of variants with each % identity, and the variantidentification.

TABLE 24 % Identity to PtIP-83Aa (SEQ ID NO: 1) Variant PolynucleotidePolypeptide 76 S04359885 SEQ ID NO: 428 SEQ ID NO: 518 76 S04359888 SEQID NO: 429 SEQ ID NO: 519 73 S04359896 SEQ ID NO: 430 SEQ ID NO: 520 73S04359899 SEQ ID NO: 431 SEQ ID NO: 521 77 S04359902 SEQ ID NO: 432 SEQID NO: 522 76 S04359909 SEQ ID NO: 433 SEQ ID NO: 523 77 S04359911 SEQID NO: 434 SEQ ID NO: 524 75 S04359942 SEQ ID NO: 435 SEQ ID NO: 525 74S04359944 SEQ ID NO: 436 SEQ ID NO: 526 73 S04359948 SEQ ID NO: 437 SEQID NO: 527 81 S04359951 SEQ ID NO: 438 SEQ ID NO: 528 80 S04359988 SEQID NO: 439 SEQ ID NO: 529 81 S04359991 SEQ ID NO: 440 SEQ ID NO: 530 77S04360034 SEQ ID NO: 441 SEQ ID NO: 531 76 S04360059 SEQ ID NO: 442 SEQID NO: 532 73 S04360064 SEQ ID NO: 443 SEQ ID NO: 533 73 S04360087 SEQID NO: 444 SEQ ID NO: 534 73 S04360095 SEQ ID NO: 445 SEQ ID NO: 535 77S04360104 SEQ ID NO: 446 SEQ ID NO: 536 74 S04360110 SEQ ID NO: 447 SEQID NO: 537 77 S04360119 SEQ ID NO: 448 SEQ ID NO: 538 73 S04360122 SEQID NO: 449 SEQ ID NO: 539 75 S04360130 SEQ ID NO: 450 SEQ ID NO: 540 78S04360132 SEQ ID NO: 451 SEQ ID NO: 541 93 S04360136 SEQ ID NO: 452 SEQID NO: 542 93 S04360141 SEQ ID NO: 453 SEQ ID NO: 543 77 S04360143 SEQID NO: 454 SEQ ID NO: 544 76 S04360146 SEQ ID NO: 455 SEQ ID NO: 545 75S04360160 SEQ ID NO: 456 SEQ ID NO: 546 79 S04360435 SEQ ID NO: 457 SEQID NO: 547 77 S04360466 SEQ ID NO: 458 SEQ ID NO: 548 77 S04360467 SEQID NO: 459 SEQ ID NO: 549 73 S04360469 SEQ ID NO: 460 SEQ ID NO: 550 78S04360485 SEQ ID NO: 461 SEQ ID NO: 551 80 S04360504 SEQ ID NO: 462 SEQID NO: 552 76 S04360545 SEQ ID NO: 463 SEQ ID NO: 553 77 S04360574 SEQID NO: 464 SEQ ID NO: 554 76 S04360579 SEQ ID NO: 465 SEQ ID NO: 555 74S04360592 SEQ ID NO: 466 SEQ ID NO: 556 79 S04360619 SEQ ID NO: 467 SEQID NO: 557 77 S04360626 SEQ ID NO: 468 SEQ ID NO: 558 77 S04360660 SEQID NO: 469 SEQ ID NO: 559 78 S04360664 SEQ ID NO: 470 SEQ ID NO: 560 77S04360699 SEQ ID NO: 471 SEQ ID NO: 561 76 S04360787 SEQ ID NO: 472 SEQID NO: 562 99 S04363576 SEQ ID NO: 473 SEQ ID NO: 563 97 S04363577 SEQID NO: 474 SEQ ID NO: 564 95 S04363578 SEQ ID NO: 475 SEQ ID NO: 565 97S04363579 SEQ ID NO: 476 SEQ ID NO: 566 97 S04363580 SEQ ID NO: 477 SEQID NO: 567 95 S04363584 SEQ ID NO: 478 SEQ ID NO: 568 97 S04363585 SEQID NO: 479 SEQ ID NO: 569 96 S04363587 SEQ ID NO: 480 SEQ ID NO: 570 96S04363588 SEQ ID NO: 481 SEQ ID NO: 571 97 S04363593 SEQ ID NO: 482 SEQID NO: 572 95 S04363594 SEQ ID NO: 483 SEQ ID NO: 573 96 S04363600 SEQID NO: 484 SEQ ID NO: 574 97 S04363605 SEQ ID NO: 485 SEQ ID NO: 575 96S04363608 SEQ ID NO: 486 SEQ ID NO: 576 96 S04363609 SEQ ID NO: 487 SEQID NO: 577 97 S04363612 SEQ ID NO: 488 SEQ ID NO: 578 94 S04363619 SEQID NO: 489 SEQ ID NO: 579 97 S04363623 SEQ ID NO: 490 SEQ ID NO: 580 94S04363625 SEQ ID NO: 491 SEQ ID NO: 581 93 S04363626 SEQ ID NO: 492 SEQID NO: 582 97 S04363629 SEQ ID NO: 493 SEQ ID NO: 583 96 S04363631 SEQID NO: 494 SEQ ID NO: 584 95 S04363632 SEQ ID NO: 495 SEQ ID NO: 585 94S04363638 SEQ ID NO: 496 SEQ ID NO: 586 98 S04363643 SEQ ID NO: 497 SEQID NO: 587 97 S04363644 SEQ ID NO: 498 SEQ ID NO: 588 97 S04363646 SEQID NO: 499 SEQ ID NO: 589 95 S04363648 SEQ ID NO: 500 SEQ ID NO: 590 96S04363659 SEQ ID NO: 501 SEQ ID NO: 591 94 S04363660 SEQ ID NO: 502 SEQID NO: 592 96 S04363662 SEQ ID NO: 503 SEQ ID NO: 593 97 S04363663 SEQID NO: 504 SEQ ID NO: 594 64 S04367796 SEQ ID NO: 505 SEQ ID NO: 595 83S04367808 SEQ ID NO: 506 SEQ ID NO: 596 76 S04367849 SEQ ID NO: 507 SEQID NO: 597 78 S04367850 SEQ ID NO: 508 SEQ ID NO: 598 80 S04367851 SEQID NO: 509 SEQ ID NO: 599 78 S04367860 SEQ ID NO: 510 SEQ ID NO: 600 77S04367872 SEQ ID NO: 511 SEQ ID NO: 601 77 S04367882 SEQ ID NO: 512 SEQID NO: 602 79 S04367903 SEQ ID NO: 513 SEQ ID NO: 603 78 S04367917 SEQID NO: 514 SEQ ID NO: 604 78 S04367945 SEQ ID NO: 515 SEQ ID NO: 605 80S04367977 SEQ ID NO: 516 SEQ ID NO: 606 78 S04367983 SEQ ID NO: 517 SEQID NO: 607 87 S04371015 SEQ ID NO: 718 SEQ ID NO: 728 88 S04371039 SEQID NO: 719 SEQ ID NO: 729 84 S04371062 SEQ ID NO: 720 SEQ ID NO: 730 86S04371086 SEQ ID NO: 721 SEQ ID NO: 731 73 S04382521 SEQ ID NO: 722 SEQID NO: 732 84 S04382532 SEQ ID NO: 723 SEQ ID NO: 733 98 S04382574 SEQID NO: 724 SEQ ID NO: 734 88 S04382581 SEQ ID NO: 725 SEQ ID NO: 735 89S04382591 SEQ ID NO: 726 SEQ ID NO: 736 87 S04382601 SEQ ID NO: 727 SEQID NO: 737

TABLE 25 % Identity to PtIP-83Aa (SEQ ID NO: 1) # variants Variants 99 1S04363576 98 2 S04363643, S04382574 97 12 S04363646, S04363629,S04363612, S04363663, S04363585, S04363580, S04363623, S04363579,S04363605, S04363644, S04363593, S04363577 96 8 S04363662, S04363587,S04363631, S04363659, S04363608, S04363600, S04363588, S04363609 95 5S04363648, S04363632, S04363584, S04363594, S04363578 94 4 S04363660,S04363619, S04363625, S04363638 93 3 S04363626, S04360136, S04360141 891 S04382591 88 2 S04371039, S04382581 87 2 S04371015, S04382601 86 1S04371086 84 2 S04371062, S04382532 83 1 S04367808 81 2 S04359951,S04359991 80 4 S04360504, S04367851, S04367977, S04359988 79 3S04367903, S04360435, S04360619 78 8 S04367983, S04367945, S04367850,S04360485, S04367917, S04360664, S04360132, S04367860 77 14 S04367872,S04360467, S04360119, S04367882, S04360104, S04360626, S04359902,S04360143, S04359911, S04360034, S04360660, S04360574, S04360466,S04360699 76 9 S04359909, S04360787, S04359888, S04359885, S04360579,S04360146, S04360059, S04367849, S04360545 75 3 S04359942, S04360130,S04360160 74 3 S04360592, S04360110, S04359944 73 9 S04359896,S04360122, S04360469, S04360087, S04359899, S04360064, S04360095,S04359948, S04382521 64 1 S04367796

The above description of various illustrated embodiments of thedisclosure is not intended to be exhaustive or to limit the scope to theprecise form disclosed. While specific embodiments of and examples aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. The teachings providedherein can be applied to other purposes, other than the examplesdescribed above. Numerous modifications and variations are possible inlight of the above teachings and, therefore, are within the scope of theappended claims.

These and other changes may be made in light of the above detaileddescription. In general, in the following claims, the terms used shouldnot be construed to limit the scope to the specific embodimentsdisclosed in the specification and the claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, manuals, books or otherdisclosures) in the Background, Detailed Description, and Examples isherein incorporated by reference in their entireties.

Efforts have been made to ensure accuracy with respect to the numbersused (e.g. amounts, temperature, concentrations, etc.) but someexperimental errors and deviations should be allowed for. Unlessotherwise indicated, parts are parts by weight, molecular weight isaverage molecular weight; temperature is in degrees centigrade; andpressure is at or near atmospheric.

That which is claimed is:
 1. A polypeptide comprising a heterologoussignal sequence or transit peptide operably linked to the PtIP-83polypeptide of SEQ ID NO:
 1. 2. The polypeptide of claim 1, wherein thePtIP-83 polypeptide has insecticidal activity against Corn Earworm(Helicoverpa zea).
 3. The polypeptide of claim 1, wherein the PtIP-83polypeptide specifically binds to a brush border membrane vesicleisolated from a Lepidoptera.
 4. The polypeptide of claim 1, wherein thePtIP-83 polypeptide disrupts the ionic balance across a brush bordermembrane vesicle isolated from a Lepidoptera.
 5. A recombinantpolynucleotide encoding the PtIP-83 polypeptide of SEQ ID NO: 1, whereinthe polynucleotide is operably linked to a heterologous regulatoryelement.
 6. The recombinant polynucleotide of claim 5, wherein thepolynucleotide is a non-genomic polynucleotide.
 7. The recombinantpolynucleotide of claim 6, wherein the polynucleotide is a cDNA, asynthetic polynucleotide or has codons optimized for expression in anagriculturally important crop.
 8. A transgenic plant comprising thepolynucleotide of claim
 5. 9. A DNA construct comprising thepolynucleotide of claim 5 operably linked to a heterologous regulatoryelement.
 10. A transgenic plant or plant cell comprising the DNAconstruct of claim
 9. 11. An agricultural composition comprising aninsecticidal effective dose of the PtIP-83 polypeptide of claim
 1. 12.The agricultural composition of claim 11 further comprising at least oneagriculturally acceptable carrier.
 13. A method for controlling aninsect pest population, comprising contacting the insect pest populationwith the polypeptide of claim
 1. 14. A method of inhibiting growth orkilling an insect pest, comprising contacting the insect pest with acomposition comprising the polypeptide of claim
 1. 15. A method ofcontrolling Lepidoptera and/or Coleoptera insect infestation in atransgenic plant and providing insect resistance management, comprisingexpressing in the plant the polynucleotide of claim
 5. 16. A method forcontrolling an insect pest population, comprising contacting the insectpest population with the transgenic plant or plant cell of claim
 10. 17.A method of inhibiting growth or killing an insect pest, comprisingcontacting the insect pest with the transgenic plant or plant cell ofclaim
 10. 18. The method of any one of claim 13, 14, 15, 16 or 17,wherein the insect or insect population is resistant to at least one Bttoxin.
 19. The recombinant polynucleotide of claim 5, wherein thepolynucleotide comprises the sequence of SEQ ID NO: 2.